A regulatory receptor network directs the range and output of the Wingless signa>

The potent activity of Wnt/Wingless (Wg) signals necessitates sophisticated mechanisms that spatially and temporally regulate their distribution and range of action. The two main receptor components for Wg [Arrow (Arr) and Frizzled 2 (Fz2)] are transcriptionally downregulated by Wg signaling, thus forming gradients that oppose that of Wg. This study analyze the relevance of this transcriptional regulation for the formation of the Wg gradient in the Drosophila wing disc by combining in vivo receptor overexpression with an in silico model of Wg receptor interactions. The experiments show that ubiquitous upregulation of Arr and Fz2 has no significant effects on Wg output, whereas clonal overexpression of these receptors leads to signaling discontinuities that have detrimental phenotypic consequences. These findings are supported by an in silico model for Wg diffusion and signal transduction, which suggests that abrupt changes in receptor levels causes discontinuities in Wg signaling. Furthermore, a 200 bp regulatory element in the arr locus was identified that can account for the Arr gradient, and it was shown that this is indirectly negatively controlled by Wg activity. Finally, the role of Frizzled 3 (Fz3) in was analyzed this system, and its expression, which is induced by Wg, was found to contribute to the establishment of the Arr and Fz2 gradients through counteracting canonical signaling. Taken together, these results provide a model in which the regulatory network of Wg and the three receptor components account for the range and shape of this prototypical morphogen system (Schilling, 2014).

During the development of a metazoan organism, signaling events are precisely regulated. One frequently employed mode of regulation is feedback loops. This study analyzed a network of feedback loops in the Drosophila wing pouch that regulate receptor abundance, and thus the range of distribution and signaling output of Wg (Schilling, 2014).

Receptors sequester their ligands and, thereby, impact upon the range of the signal. A transcriptional regulatory link between receptor expression and signaling activity, causing up- or downregulation of receptor levels in cells in response to the signal, can thus restrict or extend the signaling range. For example, the Hedghog (Hh) signal induces the expression of its receptor Patched (Ptc), a regulatory link which severely narrows the Hh activity. In the case of Wg, this study observed the opposite. Wg signaling appeared to extend the range of Wg distribution by transcriptionally downregulating expression of arr and fz2; downregulation of the receptors hinders the formation of Wg-Arr-Fz2 complexes. This allows superfluous Wg to diffuse further away from the source without being immobilized by its receptors. In agreement with this notion, a slightly narrower distribution was observed of extracellular Wg in discs that expressed Fz2 or Arr under the tubulinα1 promoter. Quantifying these observations in discs that compartmentally overexpressed the receptor, a subtle reduction of the decay length (corresponding to a slightly steeper Wg distribution) was observed in compartments that overexpressed the receptor compared with that in wild-type compartments (Schilling, 2014).

In apparent contradiction, a previous study has shown that high levels of Fz2 can stabilize Wg and promote Wg spreading; accordingly, this study observed an accumulation of Wg when repeating this experiment by overexpressing Fz2 using the GAL4-UAS system. These contradictory findings can be reconciled by taking into account the different strength of Fz2 upregulation in the two experimental setups -- Fz2 expression that is driven by the tubulinα1 promoter leads to a relatively mild upregulation of the receptor by, approximately, a factor of 2, whereas overexpression by using the GAL4-UAS system causes a much stronger overexpression. Presumably, Arr becomes the limiting factor in UAS-Fz2-overexpressing cells, a situation that might prevent the surplus Wg-Fz2 complexes from being internalized, thus causing an extracellular accumulation of Wg. If Fz2 is only moderately overexpressed, sufficient Arr protein might be available to allow this extra Fz2 to form Wg-Arr-Fz2 complexes, which are subsequently internalized, leading to a slight narrowing of the gradient because there is less free and diffusible Wg. Consistent with this notion, simultaneous strong overexpression of both of the receptors Fz2 and Arr, by means of Gal4, leads to a reduction of extracellular Wg levels (Schilling, 2014).

Although Wg signaling transcriptionally represses both Arr and Fz2, ubiquitous overexpression of Arr, or Fz2, had no phenotypic consequences. Unexpectedly, however, severe phenotypes arose upon mosaic expression of the tub>arr or tub>fz2 transgenes. Theoretical modeling and reporter gene analysis indicated that cells that had elevated receptor levels ectopically activated the pathway when situated close to wild-type cells. Apparently, the 'high-receptor-level state' allows tub-fz2 or tub-arr cells to engage in ligand-receptor interactions that depend on the 'low-receptor-level state' of their neighbors. One plausible explanation might be that tub-fz2, or tub-arr, cells bind to Wg that diffuses in from neighboring wild-type cells (Schilling, 2014).

The different outcome of clonal versus uniform alteration of the Wg pathway is reminiscent of observations that have been reported by Piddini and Vincent (2009), where loss of Wg signaling in the entire P compartment had no impact on the expression of low-threshold target genes but resulted in their repression, and in patterning defects, when Wg signaling was only clonally abolished. Piddini and Vincent also used different patterns of Wg receptor expression for their experiments, and they explained their findings by postulating that there is a Wg-induced, still to be identified, inhibitory signal that negatively regulates target gene expression in surrounding cells (Schilling, 2014).

In an additional layer of negative-feedback regulation in the wing pouch, Wg signaling activates the expression of the Frizzled family member Fz3. Fz3 seems to act as a negative regulator of Wg signaling by repressing Wg signaling readouts and downregulating Wg receptor levels. Various models could be envisaged of how Fz3 acts as an inhibitor of Wg signaling. As it has been demonstrated that Fz3 is able to bind Wg, Fz3 could work as a decoy receptor that acts as a molecular trap by binding to Wg without eliciting a signal. Decoy receptors are often part of negative-feedback mechanisms. In the Drosophila epidermal growth factor (EGF) system, the pathway inhibitor Argos is a target of EGF signaling and functions as a decoy receptor. In vertebrates, decoy Frizzled receptors have been identified that modulate Wnt signaling - secreted Frizzled-related molecules (sFRPs) have strong homology to the Frizzled extracellular domains. sFRPs inhibit signaling by directly binding to the Wnt ligands. No sFRP gene has been identified in the Drosophila genome (Schilling, 2014).

In another scenario, Fz3 could work as a negative regulator of Wg receptors. Its function could be analogous to that of ZNRF3 and RNF43 in crypt base columnar intestinal stem cells. These related E3 ubiquitin ligases have been shown to regulate the stability and levels of cell-surface Fz and LRP5/6, through internalization and lysosomal degradation of the receptor components in the presence of Wnt signaling. Several of the current experimental findings indicate that Fz3 might work as an inhibitor of Wg feedback at the receptor level - firstly, decreased Arr and Fz2 levels were observed in compartments that overexpressed Fz3, and secondly, Arr and Fz2 levels were increased in fz3 mutant wing discs. Most probably, Fz3 acts by more than one mechanism - cells that overexpressed Fz3 in the Wg stripe lead to Arr downregulation, whereas cells that overexpressed Fz3 outside of the Wg stripe lead to Arr upregulation. Furthermore, extracellular Wg was stabilized upon Fz3 overexpression. In a wild-type situation, this stabilization of Wg might contribute to a broader Wg gradient and promote signaling in the outskirts of the wing pouch. Taken together, these findings suggest that only Wg-bound Fz3 causes inhibition of the pathway (Schilling, 2014).

The post-translational regulation of Wg receptor levels was not the focus of this study, but substantial efforts were undertaken to further characterize the transcriptional regulation of the receptor genes. In particular, attempts were mead to identify the regulatory elements of these genes that mediated the feedback loops. The isolation of a 200 bp fragment of the arr locus and a 300 bp fragment of the fz3 gene (each of which was responsive to Wg signaling and drove reporter gene expression in a pattern that was reminiscent of the endogenous expression pattern) allowing an investigation of whether the Wg pathway controls these genes directly or indirectly; fz3 appeared to be a direct target of canonical Wg signaling, whereas arr did not. Pan-binding sites were dispensable in the minimal arr enhancer, indicating that either Arm regulates the transcriptional activity of arr through another DNA-binding protein, or that Arm and/or Pan transcriptionally induce one (or more) negative regulators that, in turn, regulate arr expression. Hence, although the Wg pathway has been reported to possess the capacity to directly negatively regulate transcription, it apparently does not use this mechanism to attenuate arr expression (Schilling, 2014).

Including transcriptional Wg receptor downregulation in the model led to a broader distribution of Wg - receptor downregulation by ligand-induced endocytosis consumes the ligand, this was not the case for transcriptional repression. The broadening of the Wg distribution area under a mechanism of transcriptional receptor repression might facilitate a robust signaling readout for high-threshold Wg target genes (Schilling, 2014).

A recent study indicates that the long-range Wg gradient might be less important for imaginal disc patterning than assumed previously. Hence, it is also conceivable that the receptor gradients are not essential, a notion supported by the finding that uniform misexpression of Arr or Fz2 in the wing imaginal disc had no phenotypic consequences. Nevertheless, it remains to be determined whether the Arr and Fz2 gradients are dispensable; the tubArr transgene is not able to rescue arr loss-of-function mutants (Schilling, 2014).

So far, most quantitative models of the Wnt-Wg pathway have focused on intracellular events, and only a few models have taken into account the spatial aspects of this signaling system. The model in this paper is the first to systematically study the roles of Wg-receptor complexes -- Wg-ArrFz2 and Wg-Fz3 -- in the spatial profile of Wg signaling, as well as being the first to be challenged experimentally by manipulations of the receptor levels. The cell-based modeling approach of ligand receptor interactions allowed varying of all parameters in a cell-autonomous manner, which has not been done in previous studies. This technique is, thus, an ideal tool to predict the impact of clonal conditions with cellular precision, which have historically formed the basis of experimental approaches in Drosophila but have also become increasingly available in vertebrates (Schilling, 2014)

Endocytic trafficking of Wingless and its receptors, Arrow and DFrizzled-2, in the Drosophila wing

During animal development, Wnt/Wingless (Wg) signaling is required for the patterning of multiple tissues. While insufficient signal transduction is detrimental to normal development, ectopic activation of the pathway can be just as devastating. Thus, numerous controls exist to precisely regulate Wg signaling levels. Endocytic trafficking of pathway components has recently been proposed as one such control mechanism. This study characterizes the vesicular trafficking of Wg and its receptors, Arrow and DFrizzled-2 (DFz2), and investigates whether trafficking is important to regulate Wg signaling during dorsoventral patterning of the larval wing. A role for Arrow and DFz2 in Wg internalization has been demonstrated. Subsequently, Wg, Arrow and DFz2 are trafficked through the endocytic pathway to the lysosome, where they are degraded in a hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs)-dependent manner. Surprisingly, Wg signaling is not attenuated by lysosomal targeting in the wing disc. Rather, it is suggested that signaling is dampened intracellularly at an earlier trafficking step. This is in contrast to patterning of the embryonic epidermis, where lysosomal targeting is required to restrict the range of Wg signaling. Thus, signal modulation by endocytic routing will depend on the tissue to be patterned and the goals during that patterning event (Rives, 2006).

During patterning and growth of the wing imaginal disc, cells along the D/V axis interpret positional information and, hence, their fate, from the concentration of Wg ligand. The graded distribution of Wg, with high levels near the source at the D/V boundary and low levels toward the edges of the wing pouch, is therefore crucial for normal wing development. Lysosomal targeting of Wg and its receptors has been proposed as a mechanism for shaping the Wg gradient and attenuating signal transduction. To address this model, both trafficking to the lysosome and lysosome function was interfered with using genetic and pharmacological means (Rives, 2006).

In Drosophila, the hrs loss of function allele is a valuable tool for interrupting vesicular traffic to the lysosome. Hrs functions in late endosome invagination, a process that separates endocytic cargo to be recycled from cargo destined for the lysosome. Trafficking of the EGFR and Torso RTKs into the late endosome/MVB is an important step in signal attenuation; hrs mutant embryos experience elevated tyrosine kinase signaling due to the persistence of active receptors. Likewise, in the wing disc and the ovarian follicle cell, Hrs is required for downregulation of Tkv levels and dampening of the Dpp signal. Thus, Hrs activity is required to attenuate multiple developmental signals (Rives, 2006).

The fact that RTK and Dpp signaling levels are elevated in hrs mutant cells implies that active receptor complexes continue to signal inside the cell from an endocytic compartment. Although receptor internalization may turn off signaling by preventing ligand-receptor interaction, it is clear that many receptors remain active on endosomal membranes. For instance, activated EGFR can be detected in association with downstream signaling effectors on early endosomes, suggesting that signaling persists after endocytosis. This study reports dramatic intracellular accumulation of Wg, Arrow, and DFz2 in hrs or deep orange (dor) mutant wing discs; dor encodes a yeast VPS homologue required for delivery of vesicular cargo to lysosomes. Similar observations have been made for Wg and for Wg and Arrow. Given this dramatic intracellular accumulation of ligand, receptors, and a signal transducer, Wg signaling levels are expected to be elevated in hrs mutant cells. However, based on antibody stains for three Wg targets, no altered Wg signaling was detected in mutant cells. This was true for large null mutant clones, induced early in development, as well as in discs from larvae bearing a null hrs allele. The attenuation of Wg signaling, therefore, appears to be regulated differently from the attenuation of RTK and Dpp signaling (Rives, 2006).

The data suggest that Wg signaling is attenuated prior to Hrs-mediated lysosomal targeting of the receptor complex. In this case, removal of hrs prevents receptor and ligand degradation but has no bearing on signal output. Following endocytosis, internalized receptor-ligand complexes may be deactivated by physical dissociation in the increasingly acidic environment as they move through the endocytic compartment or by targeting to the lysosome for degradation. A model is favored in which the active Wg receptor complex is attenuated by dissociation earlier in the endocytic pathway; perhaps, this complex is more sensitive to pH levels in early endosomes, whereas, for example, a Dpp receptor complex is only uncoupled at the lower pH of later endosomal compartments (Rives, 2006).

Alternatively, there may be residual lysosomal degradation in hrs cells sufficient to effectively terminate signaling despite the accumulation of Wg, Arrow, and DFz2. It is not certain that Hrs is obligatory in targeting endocytic cargo to the lysosome. Internalized avidin, an endocytic tracer, still localizes to a low pH compartment in hrs mutant garland cells, suggesting that some trafficking to the lysosome continues in the absence of Hrs. Perhaps, this residual trafficking is sufficient to dampen Wg signaling levels but not RTK or Dpp levels (Rives, 2006).

In contrast to the genetic removal of hrs, treatment of wing discs with the lysosomal protease inhibitors chloroquine or NH4Cl leads to expansion of SOPs, a Wg gain-of-function phenotype. While this result agrees with the previous finding that chloroquine-treated embryos generate excess smooth cuticle, indicative of enhanced Wg signaling, it is surprising that disruption of lysosome function can affect signaling. Once internalized, receptors are sorted into inner MVB vesicles, they are presumably sequestered from intracellular effectors and thereby deactivated. If mild bases, such as chloroquine, solely affect lysosomal protease function, a step subsequent to MVB sorting, this should not affect Wg signaling output in embryos or in imaginal discs. As all endocytic compartments maintain an acidic environment that is crucial to their function, it is unlikely that alkalizing agents solely inhibit the lysosome. In a caution to their use, pharmacological reagents such as chloroquine and NH4Cl almost certainly disturb earlier pH-dependent trafficking steps as well, resulting in the accumulation of active receptor complexes. It is hypothesized that chloroquine- and NH4Cl-mediated alkalization prevents the dissociation of Wg from its receptor(s), thereby resulting in prolonged signaling (Rives, 2006).

Consistent with the excess SOP specification in chloroquine- and NH4Cl-treated discs, RNAi knockdown of Rab5 in cultured cells causes an increase in Wg-dependent reporter activation. These findings suggest that Wg signaling is normally attenuated at a trafficking step after internalization from the plasma membrane, but prior to Hrs-mediated lysosomal targeting. Such findings should be interpreted cautiously, however, as S2 cells are reported to be macrophage-like, and, thus, any effects on signaling output in these cells might not compare to that in wing disc cells in vivo. Nevertheless, attempts were made to define more precisely the trafficking step involved by treating cultured S2 cells with Shi dsRNA. So far the results have been ambiguous, since two trials demonstrated increased reporter activation while two other trials exhibited no such increase. Unfortunately, due to the compromised viability of endocytosis-defective cells in the wing disc, the DRab5 or Shi cell culture results could not be varified in vivo. However, in agreement with the data, a recent report shows enhanced Wg signaling, as evidenced by accumulation of the signal transducer Armadillo, in cells expressing a temperature-sensitive dominant negative variant of Shi. The viability issue was circumvented by transiently expressing dominant negative Shi with a 3-h upshift to the non-permissive temperature. Interestingly, no change was observed in Wg target gene expression under these conditions, suggesting that cell viability becomes compromised before such changes can occur (Rives, 2006).

While no evidence was found that lysosomal targeting modulates Wg signal output in the developing wing, it is clear that Wg, Arrow, and DFz2 are trafficked to the lysosome by Hrs. Hrs contains a conserved ubiquitin-interacting motif (UIM) and binds ubiquitin in vitro, suggesting that it regulates MVB sorting via direct interaction with ubiquitinated receptors. Monoubiquitination of cell surface receptors is emerging as an important signal for internalization and lysosomal sorting. It will be of interest to determine whether Arrow, Fz, and DFz2 undergo signaling-dependent monoubiquitination, and whether this has a consequence for Wg signaling output (Rives, 2006).

Signaling ligands are commonly internalized by receptor-mediated endocytosis, during which a ligand–receptor complex accumulates in coated pits on the plasma membrane and enters the cell in clathrin-coated vesicles. In the embryonic epidermis, endocytosis of Wg is thought to be receptor-mediated; expression of DFz2-GPI, which presumably lacks an endocytic signal, binds Wg but does not cause internalization. A similar model is predicted in the wing imaginal disc, where expression of DFz2-GPI stabilizes Wg to a greater extent than full length DFz2, most likely due to an inability to internalize Wg. Consistent with these views, it was found that extracellular Wg accumulates on the surfaces of arrow and fzdfz2 mutant cells. This striking accumulation cannot be explained by ectopic wg gene expression and likely results from impaired Wg internalization. In support of this conclusion, Wg and Arrow can colocalize in endosomes. It was still possible to detect residual Wg internalization into arrow mutant cells and fzdfz2 mutant cells. Yet, given the striking excess of extracellular Wg on receptor-deficient cells, a large increase was expected in the number of intracellular Wg puncta if Wg is internalized at a normal rate. This was not observed and led to a suggestion that Arrow, Fz, and DFz2 function as endocytic receptors for Wg. Since Fz does not contain an obvious endocytic signal, it is presumed that Arrow and DFz2 play more prominent roles. The residual intracellular Wg in receptor-deficient cells might be explained by a functional redundancy of Arrow and DFz2 in ligand internalization, in which case an absolute defect could only be observed by producing arrow-dfz2 doubly mutant cells. While this manuscript was in preparation, Piddini (2005) also reported that both DFz2 and Arrow contribute to Wg trafficking and degradation. A model was proposed in which DFz2 is important for Wg binding and internalization, while Arrow targets the Wg-DFz2 complex for degradation in the lysosome (Rives, 2006).

Contrary to hypothesis, recent evidence suggests that the accumulation of extracellular Wg on arrow and fzdfz2 mutant clones is due to upregulation of the glypican Dally-like protein (Dlp) (Han, 2005). That study also observed an increase in the level of extracellular Wg on arrow and fzdfz2 mutant clones. However, Wg accumulation was reduced if the mutant cells were compromised for the ability to make HSPGs by additional removal of sulfateless (sfl), an enzyme required for heparan sulfate biosynthesis, or brother of tout-velu (botv), a heparan sulfate copolymerase required for HSPG biosynthesis. This suggests that some of the build-up of extracellular Wg is due to trapping by excess HSPGs, rather than to a defect in endocytic trafficking (Rives, 2006).

In the process of evaluating endocytosis-defective cells for changes in Wg signaling levels, cells were frequently observed undergoing apoptosis. This is not surprising, since endocytosis is an important means for the cell to acquire macromolecules essential for viability as well as to gauge the growth needs of the tissue in which it resides. The results are troubling, though, given the widespread use of shits, DRab5DN and ShiDN in the Drosophila community. Thus, it is necessary to monitor cell viability and assay for expression of control genes when using these reagents in order to draw accurate conclusions about signaling levels (Rives, 2006).

One notable question that was not addressed experimentally is whether endocytosis of Arrow or DFz2 is induced by Wg stimulation or proceeds continuously, independent of ligand. Some evidence for Wg-induced endocytosis of DFz2 has recently been presented (Piddini, 2005). Signal-induced endocytosis is well established, especially for RTK signaling, and plays an important role in controlling signal duration. Constitutive endocytosis and recycling provide a more general means of regulating receptor concentration at the cell surface but may also be used to downregulate signaling by clearing activated receptors, as suggested for the Tkv receptor in the developing wing. Future investigation of this issue will provide insight into the regulation of Wg signaling by endocytosis (Rives, 2006).

Protein Interactions

The Wnt family of secreted glycoproteins mediate cell- cell interactions during cell growth and differentiation in both embryos and adults. Canonical Wnt signalling by way of the ß-catenin pathway is transduced by two receptor families. Frizzled proteins and lipoprotein-receptor-related proteins 5 and 6 (LRP5/6) bind Wnts and transmit their signal by stabilizing intracellular ß-catenin. Wnt/ß-catenin signalling is inhibited by the secreted protein Dickkopf1 (Dkk1), a member of a multigene family, which induces head formation in amphibian embryos. Dkk1 has been shown to inhibit Wnt signalling by binding to and antagonizing LRP5/6. The transmembrane proteins Kremen1 and Kremen2 are high-affinity Dkk1 receptors that functionally cooperate with Dkk1 to block Wnt/ß-catenin signalling. Kremen2 forms a ternary complex with Dkk1 and LRP6, and induces rapid endocytosis and removal of the Wnt receptor LRP6 from the plasma membrane. The results indicate that Kremen1 and Kremen2 are components of a membrane complex modulating canonical Wnt signalling through LRP6 in vertebrates (Mao, 2002).

Drosophila has neither dkk nor krm but rather an LRP6 homolog, arrow, which functions in Wnt signalling. To determine if they could inhibit Wnt signalling in the fly, Xenopus dkk1 and mouse krm2 were expressed as heterologous transgenes in Drosophila and the GAL4/UAS system was used with a scalloped (sd)-GAL4 driver to direct their expression to the wing disc. Development of the wing critically depends on Wnt signalling, and interference with wingless or components of the Wnt pathway characteristically results in loss of wing structures. Even though Dkk1 protein is produced in transgenic flies, it does not affect wing development by itself. However, coexpression of dkk1 and krm2 results in almost complete loss of wings, whereas expression of krm2 alone has no effect. These results indicate that Krm is required for inhibition of Wnt signalling by Dkk1, presumably by interacting with arrow. Indeed, Dkk1 binds to and functionally interacts with Drosophila arrow transfected in 293T cells. Furthermore, inhibition of krm1 and krm2 by antisense Morpholino oligonucleotides reveals that they are required for Wnt inhibition during embryonic head formation and interact with dkk1 in Xenopus. These results suggests a model whereby Dkk1 inhibits Wnt signalling by acting in concert with its receptor Kremen to form a ternary complex with LRP6, which is rapidly endocytosed. This eliminates the Wnt receptor from the plasma membrane, thus preventing Wnt–LRP6 interaction (Mao, 2002).

Activation of the Wnt signaling cascade provides key signals during development and in disease. By designing a Wnt receptor with ligand-independent signaling activity, evidence is provided that physical proximity of Arrow (LRP) to the Wnt receptor Frizzled-2 triggers the intracellular signaling cascade. A branch of the Wnt pathway has been uncovered in which Armadillo activity is regulated concomitantly with the levels of Axin protein. The intracellular pathway bypasses Gsk3ß/Zw3, the kinase normally required for controlling ß-catenin/Armadillo levels, suggesting that modulated degradation of Armadillo is not required for Wnt signaling. It is proposed that Arrow (LRP) recruits Axin to the membrane, and that this interaction leads to Axin degradation. As a consequence, Armadillo is no longer bound by Axin, resulting in nuclear signaling by Armadillo (Tolwinski, 2003).

The data argue for a different regulatory mechanism of Wg signal transduction, proceeding through the inhibition of the protein Axin, rather than through the inhibition of Zw3/GSK3β. Axin has been identified in both vertebrates and invertebrates as a negative component of the pathway. Later work established Axin as a critical scaffold protein required for the assembly and function of the degradation complex. This complex functions in the destruction of Arm/β-catenin by bringing the kinase Zw3 and Arm into close proximity, leading to the phosphorylation of Arm, and thereby targeting it to the proteasome for degradation. For efficient Arm degradation, both Axin and APC must be present in the complex. How Wg input controls activity of the degradation complex has never been properly established, although most models have focused on the inhibition of the kinase Zw3. It is also unclear whether Arm degradation always plays a central role in converting Wnt input into transcriptional responses. In sea urchins and mammals, the most obvious response to Wnt signaling is a relocalization of Arm protein from the cytoplasm to the nucleus; it has been shown that both Axin and APC have a profound effect on Arm localization that cannot be explained by their interaction with Zw3 or the degradation complex alone (Tolwinski, 2003).

Evidence is presented that the Wg signal can be transmitted through a posttranslational regulation of Axin accumulation. Despite uniform transcription of Axin using the UAS/GAL4 system, Axin accumulates to different levels in different cells across each parasegment. Cells with lower steady-state levels of Axin are those exposed to Wg input, and this was strictly dependent on Wg. Loss of Wg causes excess accumulation of Axin, whereas uniform Wg expression (and therefore signaling) lowers total Axin levels. The phenomena observed in embryos parallel earlier reports showing that Axin accumulation is affected by Wnt signaling in tissue culture cells. GSK3β phosphorylation of Axin leads to its stabilization. However, the actual role that phosphorylation plays appears to be more complex, since further work contradicted this finding. In the current experiments, the phosphorylation state of Axin was not examined in cells responding to Wg (those with low Axin levels), nor in those not exposed to Wg (high Axin levels). Therefore, whether modification may inactivate Axin or whether modification leads to removal of Axin by degradation cannot be distinguished. It was found, however, that Zw3 kinase activity is not necessary for the reduction in Axin accumulation that is observed; the Axin striping pattern is maintained in embryos that lack Zw3 function. These results argue for a link between Wg signaling and Axin accumulation that is independent of the Zw3-mediated degradation complex (Tolwinski, 2003).

Although Zw3 does not appear to be required for Axin degradation, the more upstream component, Arrow, appears to be important for this mode of Wg signal transduction. The cytoplasmic domain of Arrow interacts with Axin in the yeast two-hybrid system, an interaction also identified for one of the mammalian LRPs, mLRP5, whose rapid binding of mAxin is ligand stimulated. Binding data for Arrow are largely in agreement with the mammalian study, except that no contribution is found of the Zw3 binding region of Axin in binding of Arrow bait. Interestingly, full-length Axin fails to interact significantly with the Arrow C terminus in yeast and all the Axin clones isolated in the library screen lack sequences N-terminal to position 353. This finding suggests that an inhibitory domain is present in Axin, N-terminal to the Zw3 binding domain, and that this inhibitory domain prevents Axin from binding Arrow. It is possible that the Wnt signal necessary for the mouse Axin interaction with LRP5 induces a conformational change in Axin that removes, modifies, or otherwise displaces the inhibitory domain. In contrast, Armadillo bait significantly binds both full-length and truncated Axin. These data taken together with the demonstration in Drosophila that signaling leads to loss in Axin striping and a lowered steady-state level of Axin, suggest that the Arrow/LRP5 interaction with Axin induces a change in activity and/or stability of Axin (Tolwinski, 2003).

The prevailing view on Wnt reception states that Arrow/LRP5,6 function as coreceptors together with Frizzled proteins. It is well established that Frizzled proteins bind Wnt ligands and that this interaction is essential for Wnt signal transduction. Initial work on LRP6 extended this model in suggesting that Wnt provides a bridging function in assembling a complex of Frizzleds and Arrow, at least for the particular combination mFz8/mWnt-1/mLRP6. However, biochemically, such complex formation has also not always been confirmed. In addition, the functional significance for signaling of the observed ternary complex has not been demonstrated in vivo. Therefore an experiment was designed that tested whether, in vivo, physical proximity of Arrow and Frizzled-2 is sufficient for signaling. In fact, it was found that Frizzled-2 can initiate ligand-independent signal transduction. The constitutive activity of the Fz2-Arr[intra]chimeric protein is significant, since only expression of the fusion protein but not expression of the individual components (Fz2 and Arr[intra]) activate signal transduction. It is inferred that association of Frizzled2 with the Arrow C terminus is indeed a key step in signal initiation in vivo, and that the proximity afforded here by protein fusion also occurs during normal signaling. The Fz2-Arr[intra] chimera is uncoupled from the need for ligand to trigger the intracellular signal transduction cascade. Therefore, whether the Arrow extracellular domain participates in a true 'reception' complex with Fz2 in Wg binding cannot be addressed. Nevertheless, Arrow, or at least its C terminus, likely interacts intimately with Fz2 during signal initiation at the membrane. In addition, activation of the pathway by the Fz2-Arr[intra] chimera proceeds through canonical pathway components, most notably requiring Disheveled, a result consistent with the finding that Dsh functions downstream of Arrow. In cultured vertebrate cells, one report has suggested that in some circumstances, LRP6 can induce Wnt signal transduction independently of Disheveled. In contrast, the experiment of overexpressing biologically active Arrow cytoplasmic sequences in the form of the Fz2-Arr[intra] chimera revealed a strict Dsh dependence, suggesting instead an obligate role for the Dsh protein at signal initiation by Arrow, and by extension, presumably by vertebrate LRP5,6 (Tolwinski, 2003).

Though the Fz2-Arr[intra] chimera clearly signals, it is not as active as a Wg-stimulated endogenous receptor complex. Presumably, and not surprisingly, the protein fusion will present a distorted topology to cofactors required in the signal initiation complex, and therefore is not optimally configured for initiating signal transduction. This may explain why the chimera retains some measure of reliance on endogenous Arrow, as is apparent from a reduced level of signaling in its absence (Tolwinski, 2003).

In summary, Arrow and the Frizzled family of Wnt receptors function in a protein complex that triggers the intracellular signaling cascade. By binding to and causing a reduction in steady-state levels of Axin, Arrow provides a pivotal link between the receptor complex on the cell surface and the downstream events that control Arm activity. One consequence of Axin degradation may reflect its role as a scaffold for Zw3-mediated degradation of Arm. However, because zw3- embryos still respond to Wg input though they fail to degrade Arm, regulation of the degradation complex cannot be the only target of Wg signaling. A Zw3-independent branch in the Wg pathway is proposed, one that might regulate the release of Armadillo from Axin, resulting in nuclear accumulation and signaling (Tolwinski, 2003).

Protein Interactions: Boca, an endoplasmic reticulum protein required for trafficking of Arrow

The maturation of cell surface receptors through the secretory pathway often requires chaperones that aid in protein folding and trafficking from one organelle to another. boca is an evolutionarily conserved gene in Drosophila melanogaster, that encodes an endoplasmic reticulum protein that is specifically required for the intracellular trafficking of members of the low-density lipoprotein family of receptors (LDLRs). Two LDLRs in flies require boca function: (1) Arrow, which is required for Wingless signal transduction, and (2) Yolkless (Yl), which is required for yolk protein uptake during oogenesis. Consequently, boca is an essential component of the Wingless pathway but is more generally required for the activities of multiple LDL receptor family members (Culi, 2003).

boca was initially isolated in a combined yeast two-hybrid and double-stranded RNA interference (RNAi) screen to search for novel developmental phenotypes in Drosophila. The sequence of a boca cDNA predicts a protein of 180 amino acids. The putative Boca protein shares ~45% identity/~65%-85% similarity with open reading frames predicted in nematode, mouse [called mesoderm development (mesd)] and human genomes, suggesting that Boca has a conserved function in metazoans. The only recognizable motifs in Boca are a putative N-terminal signal sequence and a C-terminal ER retention sequence, KDEL. Consistent with the presence of KDEL, an antibody raised against Boca detects a ubiquitously expressed cytoplasmic protein. This antibody is specific for Boca because expressing double-stranded boca RNA eliminates nearly all immunoreactivity. Boca is concentrated apically in polarized cells, such as embryonic blastoderm cells. In addition, Boca colocalizes with an ER marker protein but shows no overlap with a Golgi marker or with filamentous actin (F actin) as detected by phalloidin staining (Culi, 2003).

boca is shown to be required for the activities of at least two LDL receptor family members in flies, Yl, and Arrow. This conclusion is based on obtaining both yl and arr loss-of-function phenotypes in boca mutant flies. Specifically, using clonal analysis in imaginal discs, it was found that boca mutant cells are unable to transduce a Wg signal. Epistasis experiments with other components of the Wg pathway demonstrate that boca function is required downstream of Wg and upstream of arm. Together with the observation that boca encodes an ER protein, these experiments suggested that the defect in Wg signaling is due to a block in the maturation or processing of one of the two Wg coreceptors, Fz/Fz2 or Arr. In addition, however, females with boca mutant germlines are sterile and lay eggs that have a yolkless phenotype. This phenotype is also observed when the vitellogenin receptor, encoded by the yl gene, is nonfunctional. Since both yl and arr encode members of the LDL receptor family in flies, these results suggest that the maturation of this receptor family is specifically impaired in the boca mutant. In agreement with this suggestion, the trafficking through the secretory pathway of Arr, Yl, the human LDL receptor, and a putative Drosophila lipophorin receptor (LpR2), but not Fz2, were shown to require Boca. Thus, boca is an essential component of the Wg signaling pathway but, in addition, is more broadly required for the trafficking of multiple LDL receptor family members in Drosophila (Culi, 2003).

Interestingly, boca is required for the trafficking of Yl, Arr, LpR2, and the human LDL receptor -- four dissimilar members of this receptor family. In addition to yl, arr, and the two LpR genes, there are three other LDLR family members predicted in the Drosophila genome. It seems plausible that boca is also required for the trafficking of these other LDL receptor family members in flies. The functions mediated by these receptors are currently not known and may not have been detected by these experiments. For example, these experiments would not have identified a defect in neuronal migration or lipid homeostasis. Additional experiments will therefore be required to determine if Boca plays a role in other biological processes and to definitively assess if Boca is required for the maturation of all LDLR family members in flies (Culi, 2003).

What might Boca be doing to assist in the maturation of these receptors? The results provide some clues to this question. (1) From its sequence, Boca is predicted to be a luminal ER protein and immunostaining experiments support this prediction. Specifically, in wild-type cells Boca colocalizes with an ER marker and shows no overlap with a marker for the Golgi apparatus. (2) Immunofluorescence studies in both S2 cells and imaginal discs suggest that Boca is required for the trafficking of LDLRs through the secretory pathway. These receptors appear to remain in the ER in the absence of Boca function. The boca-dependent cell surface localization of Arr-flu was confirmed by surface biotinylation experiments in S2 cells. Taken together, these results suggest that Boca is required for the transport of LDL receptor family members from the ER to the Golgi apparatus. According to this view, boca is analogous to Star, which is required for the transport from the ER to the Golgi of Spitz, a ligand for the epidermal growth factor receptor in Drosophila. Similarly, during dorsoventral patterning of the Drosophila embryo, the ER protein Windbeutel is required for the transport of Pipe, a sulfotransferase, from the ER to the Golgi (Culi, 2003).

A third observation described here is that incorrect disulfide bonds form in both Arr-flu and Yl when Boca activity is compromised. Incorrect disulfide bond formation suggests a defect in protein folding. Thus, although its precise biochemical activity is not known, these results are consistent with the idea that Boca is a molecular chaperone that assists in the folding of LDL receptor family members in the ER. Consistent with this idea, the murine homolog of Boca, Mesd, is able to weakly interact with LRP5 and LRP6 in immunoprecipitation experiments, suggesting that Mesd/Boca may be in a complex with LDLRs. In addition, when proteins are misfolded during their progression through the secretory pathway, a quality control system blocks their exit from the ER and targets them for degradation. The results are consistent with both of these responses. In S2 cells, Arr-flu, LpR2, and human LDLR accumulate in the ER in the absence of Boca, while in vivo Arr-flu and Yl are present at lower levels and mislocalize in the boca1 mutant (Culi, 2003).

Although they share some similarities, Boca is distinct from another ER protein, receptor associated protein (RAP), that assists in the trafficking of a subset of LDL receptor family members in mammalian cells. RAP binds tightly to low-density lipoprotein receptor-related protein (LRP) and blocks its ability to bind and endocytose ligands. In contrast, Boca does not bind with high affinity to Arrow. Thus, if Boca helps LDLRs to fold, it may preferentially interact with nascent or unfolded proteins as they are translated or, alternatively, may interact with LDLRs indirectly. Another potential difference between RAP and Boca is that RAP is specifically required for the function of LRP, but not other LDLRs. In contrast, Boca is required for the trafficking of several divergent members of this family in flies. Boca and RAP are also unrelated proteins and the Drosophila genome sequence predicts the existence of an uncharacterized RAP homolog. Finally, RAP knockout mice are viable and have only reduced amounts of functional LRP whereas boca is essential for viability in the fly. As in flies, the mouse homolog of boca is also essential for viability. Thus, Boca appears to play a more essential and general role in the trafficking of LDLRs than RAP. In the future, it will be important to define a more precise biochemical function for Boca and to determine if RAP and Boca function together in the trafficking of a subset of LDL receptor family members (Culi, 2003).

The phenotypes observed in boca mutants indicate that Boca is specifically required to transduce the Wg signal and for Yl function during oogenesis. No phenotypes were obtained that would indicate a defect in the other major developmental signaling pathways in flies, including the Hedgehog, Decapentaplegic, Epidermal Growth Factor, and Fibroblast Growth Factor pathways. These genetic experiments suggest that Boca is specifically required for the activities of LDLR family members. It is unusual for a chaperone to be specifically required for several divergent members of a single family. Some chaperones, such as BiP or Hsp90, are generally required for the folding of many proteins whereas others are required to assist in the folding of a very specific subset of proteins. NinaA, for example, is critical for the trafficking of the major rhodopsin (Rh1) in flies, but is not required for the maturation of other G protein coupled receptors. In the future, it will be interesting to determine which of the features shared among LDLR family members make their trafficking through the secretory pathway dependent upon Boca. Other questions raised by these results are whether other classes of cell surface receptors have dedicated chaperones and how Boca interfaces with the more generally acting ER chaperones. Finally, although Boca appears to be present in all cells, these results raise the possibility that its regulation could be a way to modulate LDLR activity (Culi, 2003).

A large number of mutations in the human LDL receptor have been described that result in hypercholesterolemia. In addition to LDL receptor mutations, alterations in an unlinked gene that is required for LDL receptor function can also result in hypercholesterolemia. The human boca homolog maps to chromosome 15q25-q26, a region that has been linked to hypercholesterolemia in some families. Although this linkage has been disputed, the current results raise the possibility that altered versions of the human boca homolog could also contribute to hypercholesterolemia in some genetically predisposed families. In the mouse, the boca homolog corresponds to one of two genes that are deleted in the lethal mutation; mesoderm development (mesd) and additional studies demonstrate that the boca ortholog in the mouse is mesd. Interestingly, homozygous mesd mice fail to form a primitive streak and, consistent with these results, appear to have a defect in Wnt signaling (Culi, 2003).

In summary, boca encodes an evolutionarily conserved ER protein that is required for the trafficking and, therefore, activity of both Arr and Yl in Drosophila. It is postulated that boca is a molecular chaperone that is specifically dedicated to assist in the folding, trafficking, and quality control of the LDLR family (Culi, 2003).

Wnt/Wingless signaling through beta-catenin requires the function of both LRP/Arrow and frizzled classes of receptors

Wnt/Wingless (Wg) signals are transduced by seven-transmembrane Frizzleds (Fzs) and the single-transmembrane LDL-receptor-related proteins 5 or 6 (LRP5/6) or Arrow. The aminotermini of LRP and Fz were reported to associate only in the presence of Wnt, implying that Wnt ligands form a trimeric complex with two different receptors. However, it was recently reported that LRPs activate the Wnt/beta-catenin pathway by binding to Axin in a Dishevelled-independent manner, while Fzs transduce Wnt signals through Dishevelled to stabilize beta-catenin. Thus, it is possible that Wnt proteins form separate complexes with Fzs and LRPs, transducing Wnt signals separately, but converging downstream in the Wnt/beta-catenin pathway. The question then arises whether both receptors are absolutely required to transduce Wnt signals. A sensitive luciferase reporter assay in Drosophila S2 cells was established to determine the level of Wg-stimulated signaling. Wg can synergize with DFz2 and function cooperatively with LRP to activate the beta-catenin/Armadillo signaling pathway. Double-strand RNA interference that disrupts the synthesis of either receptor type dramatically impairs Wg signaling activity. Importantly, the pronounced synergistic effect of adding Wg and DFz2 is dependent on Arrow and Dishevelled. The synergy requires the cysteine-rich extracellular domain of DFz2, but not its carboxyterminus. Finally, mammalian LRP6 and its activated forms, which lack most of the extracellular domain of the protein, can activate the Wg signaling pathway and cooperate with Wg and DFz2 in S2 cells. The aminoterminus of LRP/Arr is required for the synergy between Wg and DFz2. This study indicates that Wg signal transduction in S2 cells depends on the function of both LRPs and DFz2, and the results are consistent with the proposal that Wnt/Wg signals through the aminoterminal domains of its dual receptors, activating target genes through Dishevelled (Schweizer, 2003).

Axin and the Axin/Arrow-binding protein DCAP mediate glucose-glycogen metabolism

Axin was found as a negative regulator of the canonical Wnt pathway. Human LRP5 was originally found as a candidate gene of insulin dependent diabetes mellitus (IDDM), but its Drosophila homolog, Arrow, works as a co-receptor of the canonical Wnt signal. A previous paper described Drosophila Axin (Daxin)-binding SH3 protein, DCAP, a homolog of mammalian CAV family protein (Yamazaki, 2002). Among the subtypes, DCAPL3 shows significant homology with CAP, an essential component of glucose transport in insulin signal. Further binding assay revealed that DCAP binds to not only Axin but also Arrow, and Axin binds to not only GSK3beta but also Arrow. However, overexpression and RNAi experiments of DCAP do not affect the canonical Wnt pathway. As DCAP is expressed predominantly in insulin-target organs, and as RNAi of DCAP disrupts the pattern of endogenous glycogen accumulation in late stage embryos, it is suggested that DCAP is also involved in glucose transport. Moreover, early stage embryos lacking maternal Axin show significant delay of initial glycogen decomposition, and RNAi of Axin in S2 cells revealed quite increase of endogenous glycogen level as well as GSK3beta. These results suggest that Axin and DCAP mediate glucose-glycogen metabolism in embryo. In addition, the interaction among Axin, Arrow, and DCAP implies a possible cross-talk between Wnt signal and insulin signal (Yamazaki, 2003).

This study shows that DCAP binds to not only Axin but also the cytoplasmic tail of Arrow through SH3 domains. Usually, SH3 proteins mediate protein-protein interaction in signal transduction and often link different pathways for cross talking of signals. Therefore, it is possible that DCAP can connect Wnt signaling molecules to other pathways. Although it could not be proven that DCAP participates in the canonical Wnt pathway, it was shown that DCAP is also a functional homolog of mammalian CAP in insulin signal and controls proper localization of endogenous glycogen in late stage embryos. These results suggest that the glucose transport by DCAP can be affected by Wnt signaling molecules (Yamazaki, 2003).

In the case of Axin, it binds to GSK3β, a glucose-glycogen metabolism-modifying enzyme, and also binds to DCAP, one of the components for insulin-dependent glucose transport. Novel glycogen phenotypes were found in D-axin null mutants and DCAP-RNAi embryos. In addition, RNAi of Axin in S2 increases the level of endogenous glycogen. These findings mean that Axin controls glycogen level as well as GSK3β, and this function is thought to be another important function of Axin as well as inhibiting the canonical Wnt signal (Yamazaki, 2003).

The binding between DCAP and Arrow is also quite intriguing. Arrow is a coreceptor of the canonical Wnt pathway in Drosophila. However, its human homolog LRP5 was originally identified as a candidate gene for IDDM4. Axin binds to not only GSK3β and DCAP but also Arrow/LRP5. Therefore, it is also possible that Axin, Arrow, GSK3β, and DCAP work together in glucose-glycogen metabolism or insulin signal (Yamazaki, 2003).

Although LRP5 is well studied in the canonical Wnt pathway, the function in insulin signal still needs to be investigated further. In a previous report, LRP5 was shown to be expressed in β cells of pancreatic Langerhans islets. The disruption of LRP5 can inhibit glucose uptake into β cells and cause Insulin-Dependent Diabetes Mellitus (IDDM), because β cells would misjudge blood glucose as low levels and would not release insulin. Mammalian CAP is also supposed to interact with LRP5, so this interaction may give significant insights in IDDM (Yamazaki, 2003).

Moreover, recent works revealed that Drosophila insulin signal controls cell size and number in late stage embryo during development. It is after the initial canonical Wnt signal but the same period as DCAP expression. This means that insulin signal has another function other than controlling blood glucose level. Therefore, it is considered that Wnt signal and insulin signal molecules interact each other for development other than the canonical Wnt pathway or controlling blood glucose (Yamazaki, 2003).

Until now, several cross talks have been reported in Wnt signal. As GSK3β and Arrow are already known to be multifunctional molecules, it is quite reasonable that Axin is another bifunctional molecule both in Wnt signal and glucose-glycogen metabolism. Taking together these data, it is also reasonable to think that Wnt signal has a cross-talk with insulin signal. However, the role of Axin in the initial glycogen decomposition is still unclear. It is also unknown why DCAP has five different spliced forms. To study these questions will give significant insights into the signaling network of development (Yamazaki, 2003).

Wnt signals across the plasma membrane to activate the ß-catenin pathway by forming oligomers containing its receptors, Frizzled and LRP

Wnt-induced signaling via ß-catenin plays crucial roles in animal development and tumorigenesis. Both a seven-transmembrane protein in the Frizzled family and a single transmembrane protein in the LRP family (LDL-receptor-related protein 5/6 or Arrow) are essential for efficiently transducing a signal from Wnt, an extracellular ligand, to an intracellular pathway that stabilizes ß-catenin by interfering with its rate of destruction. However, the molecular mechanism by which these two types of membrane receptors synergize to transmit the Wnt signal is not known. Mutant and chimeric forms of Frizzled, LRP and Wnt proteins, small inhibitory RNAs, and assays for ß-catenin-mediated signaling and protein localization in Drosophila S2 cells and mammalian 293 cells were used to study transmission of a Wnt signal across the plasma membrane. The findings are consistent with a mechanism by which Wnt protein binds to the extracellular domains of both LRP and Frizzled receptors, forming membrane-associated hetero-oligomers that interact with both Disheveled (via the intracellular portions of Frizzled) and Axin (via the intracellular domain of LRP). This model takes into account several observations reported here: the identification of intracellular residues of Frizzled required for ß-catenin signaling and for recruitment of Dvl to the plasma membrane; evidence that Wnt3A binds to the ectodomains of LRP and Frizzled, and demonstrations that a requirement for Wnt ligand can be abrogated by chimeric receptors that allow formation of Frizzled-LRP hetero-oligomers. In addition, the ß-catenin signaling mediated by ectopic expression of LRP is not dependent on Disheveled or Wnt, but can also be augmented by oligomerization of LRP receptors (Cong, 2004).

What is the mechanism by which Frizzled transduces a Wnt signal? Mutations that disrupt the signaling activity of Frizzled also affect the ability of Frizzled to induce membrane translocation of Dvl and reduce physical interaction between Frizzled and Dvl, suggesting that a physical interaction between Frizzled and Dvl is required for the signaling activity of Frizzled. It is proposed that Frizzled might function as a docking site for Dvl in ß-catenin signaling. The results are consistent with the finding that the Lys-Thr-x-x-x-Trp motif at the C-terminal tail of Frizzled is not only required for activating ß-catenin signaling, but also for inducing Dvl membrane translocation. The PDZ domain of Dvl has been shown to directly bind to a peptide of C-terminal region of Frizzled containing the Lys-Thr-x-x-x-Trp motif, and this peptide can inhibit Wnt/ß-catenin signaling in Xenopus. However, the binding is relatively weak (Kd~10 microM). The current results suggest that multiple regions of Frizzled might be involved in the binding with Dvl and could increase the binding affinity (Cong, 2004).

The same structural elements may be required for Frizzled to function in both the planar polarity and the ß-catenin pathways, since membrane translocation of Dvl has been implicated in planar polarity signaling, and residues essential for the activity of Frizzled in ß-catenin signaling are also important for Frizzled-induced translocation of Dvl to the plasma membrane. It is possible that other proteins in the Frizzled-Dvl complex, such as LRP in ß-catenin signaling and Flamingo in planar polarity signaling, determine the signaling consequences of interaction between Frizzled and Dvl (Cong, 2004).

What is the role of LRP in transmitting the Wnt signal and what is the function of its extracellular domain of LRP for receiving the Wnt signal? An in vitro binding assay has suggested that Wnt1 is able to bind to the extracellular domain of LRP, but analogous binding was not observed in studies with Wg protein. Results from in vitro binding assays need to be treated cautiously, as the concentrations of ligands and receptors in these assays could be significantly higher than in physiological situations, and certain components normally involved in formation of the receptor complex could be missing in these assays. Therefore, functional data are necessary to address the significance of potential binding between Wnt and LRP. The extracellular domain of LRP can be functionally replaced by the extracellular domain of Frizzled, suggesting a physiological role for a direct, or indirect, interaction of Wnt with the extracellular domain of LRP (Cong, 2004).

LRP can also transmit a signal via ß-catenin without a requirement for Wnt. Advantage was taken of two commonly used inducible oligomerization strategies to demonstrate that oligomerization of LRP6 increases its signaling activity and its interaction with Axin. Interestingly, it has been shown that the second cysteine-rich domain of DKK2 stimulates ß-catenin signaling via LRP independently of Dvl. Further experiments are needed to determine whether this DKK2 fragment activates LRP by altering the oligomerization status of LRP (Cong, 2004).

A recent study has suggested that the extracellular domain of LRP might negatively regulate the signaling activity of LRP through dimerization, which can be relieved by Wnt proteins. By contrast, the current study shows that the signaling activity of LRP was markedly increased by oligomerization. The source of this discrepancy is currently unclear. However, it was found that, when overexpressed in 293 cells, full-length LRP6 is less efficiently transferred to the plasma membrane than is LRP6{Delta}N, an observation that correlates with the lower signaling activity of LRP6. In addition, upon co-expression of MESD, a chaperone of LRP, the signaling activities of LRP6 and LRP6{Delta}N become equivalent. These data suggest that the low signaling activity of full-length LRP6 is most likely due to its poor membrane localization, and strongly argue against a negative role of the extracellular domain of LRP in Wnt signaling (Cong, 2004).

Although Wnt can bind to both Frizzled and LRP, both receptors are essential for transducing the Wnt signal. It is possible that Wnt, Frizzled and LRP form one signaling complex. Alternatively, Wnt proteins might form separate complexes with Frizzled and LRP, which turn on separate signaling pathways that converge downstream (Cong, 2004).

Several lines of evidence that support the first model are provided, and the data suggest that bringing Frizzled and LRP into proximity is sufficient to trigger signaling through ß-catenin signaling. It was shown that the Wnt signaling pathway can be fully stimulated by oligomerizing Frizzled and LRP either through the intracellular region, by directly fusing the intracellular domain of LRP6 to the C terminus of human FZ5, or through the extracellular region. Furthermore, the requirement for free Wnt proteins can be bypassed by fusing Wnt to either Frizzled or LRP. These results suggest that Wnt, Frizzled and LRP form a single signaling complex, and the function of Wnt is to form a bridge between Frizzled and LRP. It is recognized, however, that Wnt-induced oligomerization of endogenous Frizzled and LRP in living cells has not been demonstrated, nor hace the physical properties of the proposed Wnt-induced oligomers been characterized (Cong, 2004).

Why is it necessary and sufficient to bring LRP and Frizzled into proximity for transducing the Wnt signal? RNA interference studies have indicated that signaling by overexpressed LRP is strictly Dvl independent, and Dvl becomes important once Wnt and Frizzled are involved. Axin is known to interact with the C terminus of LRP, and Dvl can interact with Frizzled. Presumably, once overexpressed, a high concentration of membrane LRP is able to bring endogenous Axin to the plasma membrane, based solely on its affinity with Axin, so that Axin might be inactivated or degraded. This would explain why the signaling activity of ectopically expressed LRP is Dvl independent. Under normal physiological conditions, Frizzled and Dvl might be required to translocate Axin to the membrane LRP upon Wnt signaling. Dvl might function as a molecular chaperone to deliver Axin to the Frizzled-LRP complex, based on its affinity with both Frizzled and Axin. In addition, Frizzled and Dvl might also enhance the binding affinity between LRP and Axin through promoting phosphorylation of LRP. Therefore, the Wnt-Frizzled-LRP complex might serve as a high-affinity docking site for Axin. This model is also in agreement with the recent finding that Wnt induces translocation of Axin to the membrane in a Dvl-dependent manner. Consistent with its proposed role as a shuttle, Dvl is associated with intracellular vesicles, and interacts with both actin stress fibers and microtubules (Cong, 2004).

It is proposed that Wnt stimulates the ß-catenin pathway by relocating Axin to the plasma membrane and inactivating Axin. It is still not clear whether Wnt-induced Axin membrane translocation is a prerequisite for dissociation of the ß-catenin degradation complex. Furthermore, it is unknown whether the only function of Dvl is to facilitate the transport of Axin to the plasma membrane. It is possible that Dvl also brings certain factors to Axin upon Wnt signaling and promotes inactivation of the Axin complex. Indeed, it has been suggested that in response to Wnt, Dvl can recruit Frat/GBP, a strong inhibitor of GSK3, to the Axin-GSK3-ß-catenin complex, although a requirement for Frat/GBP in Wnt signaling has not been established genetically. Furthermore, it is unclear whether inhibition of GSK3 normally plays a major role in Wnt signaling, although dominant-negative mutants of GSK3 can activate ß-catenin signaling. It has been shown that Wnt induces dephosphorylation of Axin, which might reflect inhibition of GSK3 or dissociation of the Axin-GSK3 complex. Dephosphorylated Axin appears to be less stable and binds ß-catenin less efficiently. It is currently unknown how membrane translocation of Axin is coupled to dephosphorylation and destabilization of Axin. More work will be necessary to illustrate fully the molecular mechanism by which Wnt induces the stabilization of ß-catenin (Cong, 2004).

The roles of glypicans Dally and Dally-like protein (Dlp), the Wg receptors Frizzled (Fz) and Fz2, and the Wg co-receptor Arrow (Arr) in Wg gradient formation in the wing disc

During the wing development Wingless acts as a morphogen whose concentration gradient provides positional cues for wing patterning. The molecular mechanism(s) of Wg gradient formation is not fully understood. This study systematically analyzes the roles of glypicans Dally and Dally-like protein (Dlp), the Wg receptors Frizzled (Fz) and Fz2, and the Wg co-receptor Arrow (Arr) in Wg gradient formation in the wing disc. Both Dally and Dlp are essential and have different roles in Wg gradient formation. The specificities of Dally and Dlp in Wg gradient formation are at least partially achieved by their distinct expression patterns. Surprisingly, although Fz2 has been suggested to play an essential role in Wg gradient formation by ectopic expression studies, removal of Fz2 activity does not alter the extracellular Wg gradient. Interestingly, removal of both Fz and Fz2, or Arr causes enhanced extracellular Wg levels, which mainly results from upregulated Dlp levels. It is further shown that Notum, a negative regulator of Wg signaling, downregulates Wg signaling mainly by modifying Dally. Last, it is demonstrated that Wg movement is impeded by cells mutant for both dally and dlp. Together, these new findings suggest that the Wg morphogen gradient in the wing disc is mainly controlled by combined actions of Dally and Dlp. It is proposed that Wg establishes its concentration gradient by a restricted diffusion mechanism involving Dally and Dlp in the wing disc (Han, 2005).

One important finding of this study is that removal of the Wg receptors (Fz and Fz2) and the co-receptor Arr does not lead to a loss of extracellular Wg. Fz2 has been proposed to play a major role in Wg gradient formation in the wing disc by ectopic expression studies. Although the high capacity of Fz2 in stabilizing Wg has been demonstrated, loss-of-function results clearly show that extracellular Wg levels are not reduced in clones mutant for fz2. This is apparently not due to the overlapping function of Fz, since the extracellular Wg level is enhanced, rather than reduced, in the absence of both Fz and Fz2 functions. The results argue that Fz2 is not essential for extracellular Wg gradient formation in vivo. It is important to note that in addition to Fz and Fz2, Fz3 is also expressed in the wing disc and its expression is upregulated by Wg signaling. Although Fz3 has lower affinity than Fz2 in Wg binding and acts as an attenuator of Wg signaling, its role in Wg distribution needs to be determined (Han, 2005).

It is further demonstrated that extracellular Wg is enhanced in cells mutant for fz-fz2 or arr, suggesting that Wg receptors (Fz and Fz2) and Arr shape extracellular Wg gradient by downregulating extracellular Wg levels. The data argue that this mainly results from upregulation of Dlp. Consistent with this view, the accumulated extracellular Wg can be eliminated by loss of HSPGs in sfl-fz-fz2 or arr-botv mutant clones. Importantly, it is shown that both extracellular Wg and Dlp levels are upregulated on the cell surface of clones mutant for dsh. These data provide compelling evidence that though a feedback mechanism, Wg signaling can control the Dlp levels to regulate the extracellular Wg gradient (Han, 2005).

Another alternative possibility is that enhanced Wg levels in fz-fz2 or arr clones may be caused by impaired Wg internalization. Although a significant amount of internalized Wg vesicles has been demonstrated in fz-fz2 or arr mutant clones, this possibility cannot be ruled out, since a quantitative comparison of Wg internalization between wild-type cells and fz-fz2 or arr mutant cells is difficult. Furthermore, as mentioned above, Fz3 is expressed in the wing disc and its expression is upregulated by Wg signaling. It is possible that Fz3 may mediate the internalization of Wg in the absence of Fz and Fz2 (Han, 2005).

Evidence has been presented that Wg morphogen movement is regulated by a diffusion mechanism(s) in the wing disc. Does Wg diffuse freely in the extracellular matrix/space? In this work, it is shown that Wg fails to move across a strip of cells mutant for the HSPGs Dally and Dlp. This result suggests that Wg cannot freely diffuse in the extracellular matrix. Instead, the findings are consistent with a model in which Wg movement is mediated by the HSPGs Dally and Dlp through a restricted diffusion along the cell surface. Similar mechanisms have been proposed for Hh and Dpp. In biological systems such as imaginal discs, the restraint of Wg spreading to the surface of the epithelial cell layer is important since the folding of imaginal discs, such as the leg disc, poses a problem if the Wg gradient formation were to occur out of the plane of the epithelial cell layer through free diffusion. In agreement with this view, the model proposes that Wg gradient formation depends on Wg movement through the cell surface of the disc epithelium (Han, 2005).

Arrow (LRP6) and Frizzled2 cooperate to degrade Wingless in Drosophila imaginal discs

Lysosome-mediated ligand degradation is known to shape morphogen gradients and modulate the activity of various signalling pathways. The degradation of Wingless, a Drosophila member of the Wnt family of secreted growth factors, was investigated. One of its signalling receptors, Frizzled2, stimulates Wingless internalization both in wing imaginal discs and cultured cells. However, this is not sufficient for degradation. Indeed, as shown previously, overexpression of Frizzled2 leads to Wingless stabilization in wing imaginal discs. Arrow (the Drosophila homologue of LRP5/6), another receptor involved in signal transduction, abrogates such stabilization. Evidence is provided that Arrow stimulates the targeting of Frizzled2-Wingless (but not Dally-like-Wingless) complexes to a degradative compartment. Thus, Frizzled2 alone cannot lead Wingless all the way from the plasma membrane to a degradative compartment. Overall, Frizzled2 achieves ligand capture and internalization, whereas Arrow, and perhaps downstream signalling, are essential for lysosomal targeting (Piddini, 2005).

The main conclusion of this work is that two receptors contribute distinct though overlapping trafficking activities that together lead to degradation of Wingless. Binding data support the earlier suggestion that normally Wingless is primarily captured by a Frizzled family member and that this facilitates subsequent binding to Arrow. Wingless is internalized by Frizzled2 in the absence of Arrow. This result extends and complements recent evidence that mammalian Frizzled4 is endocytosed upon stimulation by Wnt5a. Moreover, Wingless internalization in the absence of Arrow also shows that Wingless signalling is not required for endocytosis. However, in the absence of further targeting to a lysosomal compartment, endocytosis would clearly be insufficient for degradation (Piddini, 2005).

Using gain-of-function experiments, Arrow is shown to contributes to the targeting of Wingless, maybe as a complex with Frizzled2, to a degradative compartment. As expected, loss of either Arrow or Frizzled and Frizzled2 leads to extracellular accumulation of Wingless. Frizzled and Frizzled2 are clearly redundant in this respect (as in signalling) because removal of either receptor has no noticeable effect on Wingless distribution. Interestingly, large intracellular vesicles are lost in the absence of Frizzled;Frizzled 2 but not in the absence of Arrow. It is suggested that Frizzled-mediated endocytosis is sufficient to generate these large vesicles in the absence of Arrow. The fine-grained Wingless staining seen in the absence of Frizzled;Frizzled 2 could be internalized by Arrow or by another receptor, such as Dally or Dally-like. The distinct intracellular distribution of Wingless in the absence of Frizzled;Frizzled 2 when compared with that in Arrow-deficient cells is consistent with the suggestion that the two receptor classes have distinct trafficking activities (Piddini, 2005).

It is unclear at this point whether the degrading activity of Arrow is regulated by post-translational modification or by the recruitment of other factors. Either process could be impaired in ArrowDeltaC. Work in Xenopus has identified negative regulators of Wnt signalling, Kremens, which operate by triggering LRP6 endocytosis and possibly degradation. It remains to be seen whether this leads to degradation of a Wnt during frog embryogenesis. Moreover, there is no Kremen homologue (a negative regulator of Wnt signalling identified in Xenopus that operates by triggering LRP6 endocytosis and possibly degradation) encoded by the fly genome. Clearly further work will be needed to understand the genetic control of Wnt/Wingless degradation both in flies and other systems. The data provide a simple explanation of why overexpression of Frizzled2, a receptor that mediates Wingless internalization, causes Wingless stabilization. Under such experimental conditions, Arrow becomes limiting and in the absence of an effective degradation signal, Wingless accumulates (Piddini, 2005).

Because the receptors involved in Wingless degradation are those required for signalling, Wingless degradation cannot be initiated before a signalling-competent complex is assembled. Even though signalling downstream of Armadillo is not sufficient to activate the degradation of Frizzled2-Wingless complexes, it is not known yet whether downstream signalling is necessary for degradation. In the case of EGF receptor signalling, ubiquitination (the first step towards degradation of the ligand) is contingent on the tyrosine phosphorylation that accompanies receptor activation. However, in this case, a single receptor type is involved. In the case of TGFß signalling, two receptor types are required for signal transduction. Type 2 receptor is believed to capture the ligand and this is followed by the formation of a tripartite complex with type 1 receptor. Interestingly, like Arrow, type 1 receptor brings a degradation signal such that the two types of receptor cooperate to direct the ligand towards degradation and signalling pathways appropriately. Sharing of trafficking duties by distinct receptors may provide cells with increased flexibility as expression or turnover of the two receptors could be independently modulated. It may not be a coincidence that both Dpp (the fly TGF-ß) and Wingless, which can act over a relatively long distance, use two receptors for signalling and degradation. Maybe separation of capture and degradation is a feature required for long-range signalling, perhaps by allowing modulation of local relative receptor levels (Piddini, 2005).

Further work will be needed to identify the relevant trafficking signals in Arrow and Frizzled2, as well as the mechanisms that control relative receptor levels in order to obtain a full understanding of how degradation of Wingless is tuned to generate a reliable concentration gradient (Piddini, 2005).

Casein kinase 1 gamma couples Wnt receptor activation to cytoplasmic signal transduction

Signalling by Wnt proteins (Wingless in Drosophila) has diverse roles during embryonic development and in adults, and is implicated in human diseases, including cancer. LDL-receptor-related proteins 5 and 6 (LRP5 and LRP6; Arrow in Drosophila) are key receptors required for transmission of Wnt/β-catenin signalling in metazoa. Although the role of these receptors in Wnt signalling is well established, their coupling with the cytoplasmic signalling apparatus remains poorly defined. Using a protein modification screen for regulators of LRP6, the identification of Xenopus Casein kinase 1 γ (CK1γ), a membrane-bound member of the CK1 family and homolog of Drosophila Gilgamesh, is described. Gain-of-function and loss-of-function experiments show that CK1γ is both necessary and sufficient to transduce LRP6 signalling in vertebrates and Drosophila cells. In Xenopus embryos, CK1γ is required during anterio-posterior patterning to promote posteriorizing Wnt/β-catenin signalling. CK1γ is associated with LRP6, which has multiple, modular CK1 phosphorylation sites. Wnt treatment induces the rapid CK1γ-mediated phosphorylation of these sites within LRP6, which, in turn, promotes the recruitment of the scaffold protein Axin. These results reveal an evolutionarily conserved mechanism that couples Wnt receptor activation to the cytoplasmic signal transduction apparatus (Davidson, 2005).

In a human cell-culture-based small pool expression screen for proteins able to covalently modify LRP6, CK1γ, a protein which retards ('upshifts') the migration of LRP6 on SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was identified in co-transfection assays. There are three closely related isoforms of CK1γ -- CK1γ1, 2 and 3 -- that are unique within the CK1 family in carrying putative palmitoylation sites at the carboxy terminus (TKCCCFFKR), that anchor the proteins in the plasma membrane. This is unlike CK1α, δ and epsilon, which are cytoplasmic or nuclear and are involved in the direct regulation of Wnt pathway components other than LRP5 and LRP6. Indeed, LRP6 upshift is promoted by CK1γ, while CK1epsilon has no effect on the electrophoretic mobility of LRP6. The cytoplasmic domain of LRP6 is the region modified by CK1γ, as an LRP6-δN protein that lacks the amino-terminal extracellular domain (ECD) is still able to produce the higher molecular weight bands indicative of CK1γ-mediated modification, whereas an LRP6-δC protein that lacks the carboxy-terminal intracellular domain (ICD) does not. These results suggest that the C terminus of LRP6 is modified by CK1γ phosphorylation, and this was confirmed by 32P-incorporation in vivo. Moreover, modification of LRP6 by CK1γ enhances LRP6 function in Wnt reporter assays, in which LRP6 synergizes strongly with CK1γ and more weakly with CK1epsilon, while Dishevelled-1 (Dvl1) shows the inverse behaviour, consistent with the latter being directly regulated by CK1epsilon. Similar gain-of-function results were obtained in Xenopus embryos (Davidson, 2005).

To investigate the requirement of CK1γ in LRP6 signalling, two dominant-negative forms of CK1γ were designed with point mutations (K73R or D164N) in the ATPase domain, previously identified as generating specific inhibitors for CK1epsilon. Both dominant-negative forms of CK1γ inhibit the upshift, and hence phosphorylation, of LRP6 by wild-type CK1γ, but phosphorylation of Dvl1 by CK1epsilon is unaffected. Therefore, both dominant-negative forms of CK1γ specifically inhibit CK1γ. Dominant-negative CK1γ inhibits Wnt signalling in HEK293T cells, and similar loss-of-function results were obtained in Xenopus embryos; wild-type CK1γ rescues this effect in both cases. Unlike Wnt1, Wnt3a and a constitutively active LRP6-δE(1-4) protein, lacking most of the extracellular domain, β-catenin activity is unaffected by dominant-negative forms of CK1γ. This is consistent with CK1γ acting directly on LRP6 (Davidson, 2005).

Wingless (Wg) reporter assays in Drosophila SL2 cells show that the single Drosophila homologue of CK1γ, Gilgamesh (Gish), strongly synergizes with the Drosophila LRP6 homologue, Arrow. Conversely, using an interfering RNA (RNAi) loss-of-function approach, a gish double-stranded RNA that targets all eight gish transcripts blocks Wg but not Dishevelled (Dsh) signalling. This is unlike armadillo (arm) and dsh dsRNAs, which block signalling by both Wg and Dsh. These results support an evolutionarily conserved role for CK1γ in LRP6 regulation. The absence of obvious Wg phenotypes in Drosophila gish mutants may be because at least two alternatively spliced transcripts within the gene locus are intact (Davidson, 2005).

In Xenopus embryos, maternally derived ck1γ mRNA transcripts are present ubiquitously at gastrulation, and zygotic transcription starts at around the mid-neurula stage. To address the physiological role of CK1γ, Xenopus gain-of-function and loss-of-function experiments were performed. Overexpression of CK1γ in the animal pole by messenger RNA injection leads to headless embryos, whereas injection of either a ck1γ morpholino or dominant-negative ck1γK73R mRNA reduces trunk and tail structures, and induces enlarged heads and cement glands. These phenotypes match hyper- and hypo-activation of zygotic Wnt signalling, respectively. It is concluded that CK1γ is required for Wnt-mediated antagonism of Spemann's head organizer (Davidson, 2005).

Following transfection in HEK293T cells, CK1γ tagged with enhanced yellow fluorescent protein (EYFP-CK1γ) localizes to the plasma membrane, consistent with it containing a C-terminal palmitoylation site. Deleting this region (CK1γ-δC) results in the cytoplasmic localization of CK1γ, and abolishes Wnt/β-catenin signalling, although its kinase activity is unaffected. Bioluminescence resonance energy transfer (BRET) assays reveal that CK1γ and LRP6 interact in live cells, and that this interaction requires membrane localization, as it is abolished in CK1γ-δC. Co-immunoprecipitation (CoIP) confirms that LRP6 specifically interacts with CK1γ and not CK1γ-δC, and this is independent of coexpressed Axin or GSK3β. Neither BRET nor CoIP assays show differences in CK1γ-LRP6 interaction on Wnt treatment. These results indicate that CK1γ and LRP6 associate, and that this association requires the membrane localization of CK1γ (Davidson, 2005).

To identify relevant phosphorylation sites in LRP6, serial C-terminal deletions were tested for loss of CK1γ synergy in Wnt reporter assays. The LRP6-δE(1-4) backbone generates a robust Wnt response. A gradual, not abrupt, decrease in reporter activation with progressive deletions is observed, consistent with the presence of multiple activating phosphorylation sites. LRP6-δE(1-4)-δ87, which retains a single PPPSP motif, was identified as the shortest construct still activated by CK1γ (Davidson, 2005).

Deletion of Ser and/or Thr clusters in LRP6-δE(1-4)-δ87 revealed that two regions are required for cooperation with CK1γ. To test if the two regions are also sufficient for Wnt signalling, they were linked alone, or in combination, to LDLR-δN, a heterologous low-density lipoprotein (LDL) mini-receptor structurally analogous to LRP6-δN but inactive in Wnt signalling. Neither region alone (miniA or miniB) activates Wnt signalling, but they do when combined (miniC), and this effect is further enhanced by CK1γ both in HEK293T cells and Xenopus embryos (Davidson, 2005).

The region of LRP6 contained within miniC consists of a PPPSP motif flanked by two evolutionarily conserved Ser/Thr clusters that are potential CK1 phosphorylation sites. The N-terminal CK1 site (TGA(S)7TKGT) is referred to as cluster 1, and the C-terminal CK1 site (SPATERSHYT) as cluster 2 (abbreviated 1/S/2, with the Ser residue in the PPPSP motif designated by the S). To analyse the function of the two CK1 site clusters in Wnt signalling, inactivating Ala (A) and phospho-mimicking Asp (D) substitutions of all Ser/Thr residues within these clusters (shown above in bold) were tested, using the miniC receptor as a backbone. The wild-type mini-receptor (1/S/2) is constitutively active, unlike A/S/A, indicating that the CK1 sites are required for Wnt signalling. Elimination of either CK1 site alone (A/S/2 or 1/S/A) suppresses Wnt signalling. Conversely, Asp substitutions in individual (D/S/2 or 1/S/D) or both CK1 sites (D/S/D) lead to Wnt hyperactivation. Constitutive Wnt signalling by 1/S/2, D/S/2 and 1/S/D is blocked by dominant-negative CK1γ, indicating that both clusters require CK1γ for Wnt signalling. D/S/D, on the other hand, in which both CK1 sites are artificially activated, signals independently of CK1γ. Any substitution of the PPPSP site (1/A/2 or 1/D/2) inactivates Wnt signalling, indicating that this serine is essential. The results suggest that (1) clusters 1 and 2 are both physiologically relevant CK1γ phosphorylation sites that activate Wnt signalling, and (2) they require an intact PPPSP motif (Davidson, 2005).

To show direct phosphorylation of LRP6 by CK1γ, in vitro kinase assays were performed. A phospho-specific antibody (Tp1479) was raised against a CK1 site within cluster 1, which recognizes the most C-terminal phosphorylated threonine. In vitro kinase assays were performed with CK1γ using the miniC receptor carrying various internal mutations as the substrate. Following the kinase reaction and SDS-PAGE, samples were analysed for 32P-incorporation by autoradiography, and T1479 phosphorylation by immunoblotting. The wild-type mini-receptor (1/S/2) is phosphorylated by CK1γ, unlike the construct in which all three sites are inactivated (A/A/A). In agreement with cluster 1 being a CK1 phosphorylation site, 1/A/A is robustly phosphorylated. In contrast, A/S/A, containing only the intact PPPSP site, is not phosphorylated by CK1γ. Notably, cluster 2 is only weakly phosphorylated (A/A/2), but this is enhanced either by the presence of an intact PPPSP motif (A/S/2) or mimicking cluster 1 and PPPSP phosphorylation (D/D/2). Taken together, these results indicate that clusters 1 and 2, but not the PPPSP motif, are direct target sites for CK1γ phosphorylation, and that efficient phosphorylation of cluster 2 requires N-terminal priming from the PPPSP site. T1479 is phosphorylated in all constructs containing an unmodified cluster 1, indicating that it is a bona fide target for phosphorylation by CK1γ (Davidson, 2005).

An important consequence of Wnt activation is the sequestration of Axin, a negative regulator of β-catenin, by LRP6. Therefore, it was asked whether CK1γ is required for Axin recruitment. In CoIP assays, dominant-negative CK1γ markedly reduces binding of Axin to LRP6. It has been shown that the PPPSP site of LRP6 is required for Axin binding. To determine if the two CK1 phosphorylation site clusters in LRP6 also play a role in Axin binding, CoIPs were performed using 1/S/2 site substitutions within the LRP6-δE(1-4)-δ87 protein. While only weak interaction is seen with the wild-type construct (1/S/2), mimicking CK1γ phosphorylation of cluster 1 in D/S/2 leads to robust binding of Axin. Increased binding is also observed in 1/S/D, supporting the notion that phosphorylation of cluster 2 is physiologically relevant. However, activation of this cluster by itself (A/S/D) fails to mediate Axin binding. Likewise, D/S/2 shows stronger binding than D/S/A, arguing that both CK1 sites act synergistically to promote Axin binding when they are phosphorylated. Of note, 1/D/D fails to bind Axin, indicating that a phospho-serine at the PPPSP site cannot be mimicked by an Asp, explaining the inactivity of 1/D/2. It is concluded that CK1γ is required for Axin binding, and that both of the identified CK1 phosphorylation site clusters promote this interaction (Davidson, 2005).

Wnt signalling promotes Axin binding to LRP6, and the data indicate that this is mediated by CK1γ phosphorylation at conserved sites in LRP6. An important prediction, therefore, is that Wnt stimulation leads to phosphorylation of LRP6 at sites of CK1γ phosphorylation. Indeed, CK1γ overexpression in HEK293T cells induces phosphorylation at T1479 (in cluster 1), and this can be mimicked by Wnt treatment. This Wnt-induced T1479 phosphorylation requires CK1γ, as it is blocked by the dominant-negative mutant CK1γK73R. The PPPSP motifs are also thought to become phosphorylated following Wnt stimulation. However, using a phospho-specific anti-PPPSpP antibody (Sp1490), no significant phosphorylation increase is detected after either CK1γ overexpression or Wnt treatment. Strikingly, as little as 10 min of Wnt stimulation is sufficient to induce robust T1479 phosphorylation of endogenous LRP6 in mouse embryonic fibroblasts (MEFs), P19 and HeLa cells. Unlike T1479, S1490 (in the PPPSP motif) is constitutively phosphorylated in all cell lines tested, and shows either no or weaker Wnt induction. Using a phospho-independent T1479 antibody, which recognizes total LRP6, a progressive mobility upshift is observed, which saturates after 60 min of Wnt treatment. It is concluded that Wnt stimulation results in phosphorylation of T1479 (cluster1) in a CK1γ-dependent manner (Davidson, 2005).

It has been suggested that the ECD of LRP6 exerts an autoinhibitory effect that is relieved upon Wnt stimulation, based on the observation that its deletion leads to constitutive receptor activation. Alternatively, it was suggested that deletion of the ECD enhances transport to the plasma membrane, because co-transfection of the chaperone MESD (for mesoderm development) with full-length LRP6 activates Wnt signalling as effectively as the ECD-deleted form. To distinguish between these two possibilities either full-length LRP6 and MESD or LRP6-δE(1-4) were coexpressed alone in HEK293T cells, and T1479 phosphorylation was analysed. No signal is detected in full-length LRP6 despite overexpressing MESD, while there is a strong signal in LRP6-δE(1-4). A slight increase in S1490 (PPPSP) phosphorylation was also observed in LRP6-δE(1-4) compared to full-length LRP6. These results therefore support the idea of an autoinhibitory role of the LRP6 ECD, and indicate that one of its functions is to prevent phosphorylation by CK1γ (Davidson, 2005).

In summary, this investigation indicates that CK1γ couples the extracellular Wnt signal and the cytoplasmic signal transduction machinery, and suggests a model where the net effect of CK1γ phosphorylation of LRP6 is Axin recruitment. The cytoplasmic domain of LRP6 contains five reiterated PPPSP motifs that are necessary for Wnt/β-catenin signalling. While the PPPSP motifs are phosphorylated, the data demonstrate that this is not by CK1γ, but by an unknown, probably proline-directed, kinase. As Wnt was reported to stimulate PPPSP phosphorylation, it was surprising to find not only that there is constitutive S1490 (PPPSP) phosphorylation in unstimulated cells, but that Wnt stimulates no or only weak induction at this site. Since CK1 members act on proteins previously phosphorylated by other kinases, constitutive S1490 (PPPSP) phosphorylation may prime CK1γ-mediated phosphorylation. This may well apply to all PPPSP sites in LRP6, as all are succeeded by at least one adjacent CK1 site. It is therefore proposed that the LRP6 cytoplasmic domain is modular, and each element consists of a CK1γ phosphorylation site that is primed by a phosphorylated PPPSP motif (Davidson, 2005).

Regulation of wingless signaling by the CKI family in Drosophila limb development

The Wingless (Wg)/Wnt signaling pathway regulates a myriad of developmental processes and its malfunction leads to human disorders including cancer. Recent studies suggest that casein kinase I (CKI) family members play pivotal roles in the Wg/Wnt pathway. However, genetic evidence for the involvement of CKI family members in physiological Wg/Wnt signaling events is lacking. In addition, there are conflicting reports regarding whether a given CKI family member functions as a positive or negative regulator of the pathway. This study examined the roles of seven CKI family members in Wg signaling during Drosophila limb development. Increased CKIepsilon stimulates whereas dominant-negative or a null CKIepsilon mutation inhibits Wg signaling. In contrast, inactivation of CKIalpha by RNA interference (RNAi) leads to ectopic Wg signaling. Interestingly, hypomorphic CKIepsilon mutations synergize with CKIalpha RNAi to induce ectopic Wg signaling, revealing a negative role for CKIepsilon. Conversely, CKIalpha RNAi enhances the loss-of-Wg phenotypes caused by CKIepsilon null mutation, suggesting a positive role for CKIalpha. While none of the other five CKI isoforms can substitute for CKIalpha in its inhibitory role in the Wg pathway, several CKI isoforms including CG12147 exhibit a positive role based on overexpression. Moreover, loss of Gilgamesh (Gish)/CKIgamma attenuates Wg signaling activity. Finally, evidence is provided that several CKI isoforms including CKIalpha and Gish/CKIgamma can phosphorylate the Wg coreceptor Arrow (Arr), which may account, at least in part, for their positive roles in the Wg pathway (Zhang, 2006).

The Wnt family of secreted growth factors controls many key developmental processes, including cell proliferation, cell fate determination, tissue patterning, and planar cell polarity in a wide variety of organisms. Mutations in Wnt signaling components lead to many types of cancers including colon and skin cancers. The Drosophila Wingless (Wg), a founding member of the Wnt family, controls embryonic segmental polarity and patterning of adult appendages such as wing, leg, and eye. Wg exerts its biological influence through the canonical Wnt/β-catenin pathway, which is evolutionarily conserved from invertebrates to vertebrates (Zhang, 2006).

Genetic and biochemical studies in several organisms have suggested a model for Wnt/Wg signal transduction. Binding of Wnt/Wg proteins to their cognate receptors, members of the Frizzled (Fz) family of seven transmembrane proteins, and co-receptors, LRP5/6/Arrow (Arr), activates a cytoplasmic signaling component Dishevelled (Dsh), which counteracts the activity of a destruction complex composed of Axin, APC, and the Ser/Thr kinase GSK3β/Shaggy (Sgg)/Zest White 3 (Zw3), leading to the accumulation and nuclear translocation of the transcriptional effector β-catenin/Armadello (Arm). β-catenin/Arm forms a complex with the DNA binding protein Lef1/TCF to activate Wnt/Wg target genes (Zhang, 2006).

A cohort of studies have provided evidence that CKI family members participate in many aspects of the Wnt/Wg signaling pathway (Price, 2006). CKIε was first identified as a positive regulator of the canonical Wnt pathway. Overexpression of CKIε in Xenopus embryos induced ectopic dorsal axis formation, activated Wnt-responsive genes, and rescued the axial formation of UV treated embryos. Dominant negative forms of CKIε and a pharmacological inhibitor of CKI blocked the responses to ectopic Wnt signaling in Xenopus. Biochemical and epistasis study suggested that CKIε binds Dsh and acts between Dsh and GSK3β. In vivo and In vitro kinase assays showed that CKIε can phosphorylate Dsh and a pharmacological CKI inhibitor can block Wnt induced Dsh phosphorylation, suggesting that Dsh is a target of CKIε. However, the role of CKIε appears to be more complex than it was originally anticipated. For example, it has also been shown that CKIε interacts with Axin, and Axin-bound CKIε phosphorylates APC and modulates its ability to regulate β-catenin. What makes the picture even more complicated is the finding that, in a reconstituted system of Xenopus extracts, CKIε can phosphorylate Tcf3 and enhance Tcf3-β-catenin association and β-catenin stability, implying that CKIε may also exert a positive influence downstream of GSK3β (Zhang, 2006 and references therein).

The potential role of other CKI isoforms in Wnt signaling has also been examined in several systems. In an overexpression study using Xenopus embryonic explants, all other CKI isoforms, including α, β, γ, and δ, can activate Wnt signaling (McKay, 2001). All of these CKI isoforms with the exception of CKIγ can stimulate Dsh phosphorylation in cultured cells. However, subsequent studies provided evidence that CKIα plays a negative role in Wnt/Wg signaling that acts as a priming kinase for GSK3β-mediated phosphorylation of β-catenin/Arm. Purification of the Axin-bound kinases that can prime GSK3β-mediated phosphorylation of β-catenin identified CKIα. RNAi knockdown of CKIα inhibited phosphorylation at Ser45 of β-catenin and subsequent phosphorylation by GSK3β, resulting in β-catenin stabilization. Consistent with the vertebrate results, CKIα RNAi of Drosophila embryos resulted in 'naked cuticle', a phenotype consistent with gain-of-Wg signaling (Liu, 2002). The possible role of CKIε as a priming kinase for β-catenin remained unclear. Overexpression of a dominant negative CKIε inhibited Axin-induced phosphorylation at Ser45 of β-catenin in 293 cells. In addition, RNAi knockdown of CKIε stabilized Arm in Drosophila S2+ cells, although the effect was less dramatic than CKIα RNAi knockdown. In contrast, RNAi knockdown of CKIε in 293T cells had no detectable effect on Ser45 phosphorylation and stability of β-catenin. It remains possible that CKIε plays a minor partially redundant role in β-catenin/Arm phosphorylation and the effect of its inactivation on β-catenin/Arm phosphorylation and degradation could have been masked by CKIα (Zhang, 2006 and references therein).

Although CKIα RNAi in Drosophila embryos resulted in phenotypes consistent with 'gain-of-Wg' function, the recent finding that CKIα is also a negative regulator of the Hh pathway complicated the interpretation. Because Wg and Hh cross-regulate each other during embryonic development, the 'gain-of-Wg' phenotype resulted from CKIα RNAi could be attributed to ectopic Hh signaling. To further investigate the physiological roles of the CKI family members in Wg signaling In vivo, overexpression, dominant-negative, genetic mutations, and RNAi approaches were applied to study the function of CKIε, CKIα and Gish/CKIγ in Drosophila wing development where Wg signaling is independent of Hh. The potential roles of other CKI family members were also assessed (Zhang, 2006).

This study provides the first genetic evidence that DBT/CKIε plays a pivotal positive role in the Wg pathway and provides evidence that DBT/CKIε exerts its positive influence both upstream and downstream of GSK3β. Moreover, the first genetic evidence is provided that DBT/CKIε has a negative role in addition to its predominantly positive role in the Wg pathway. Using RNAi, evidence that CKIα is the major CKI isoform that negatively regulates Wg signaling in Drosophila wing development. In addition, evidence is provided that CKIα may also have an unappreciated positive role and this could be achieved, at least in part, at the level of Arr phosphorylation. Finally, genetic evidence is provided that Gish/CKIγ has a positive role in the Wg pathway. Consistent with this finding, a recent study showed that RNAi knockdown of Gish in cultured cells reduced Wg-stimulated luciferase reporter gene expression (Davidson, 2005). In addition, Gish/CKIγ, like its vertebrate counterpart, was found to be mainly localized on the cell surface, and can effectively phosphorylate Arr, which may account for its positive role in the Wg pathway (Zhang, 2006).

CKIε was initially identified as a positive regulator in the Wnt pathway based on overexpression studies. Indeed, overexpression of XCKIε in Drosophila limb caused cell autonomous accumulation of Arm and activation of Wg responsive genes, leading to pattern abnormality consistent with ectopic Wg signaling. Although DBT/CKIε shares over 85% amino acid sequence identity with XCKIε in the kinase domain, overexpression of DBT or its kinase domain didn't induce ectopic Wg signaling. Nevertheless, overexpression of DBT induced ectopic Wg signaling in a sensitized genetic background (Zhang, 2006).

Despite the fact that CKIε has been implicated as a positive regulator of the Wnt/Wg pathway, no genetic evidence for such a role has ever been obtained until now. One reason could be that CKIε participates in multiple cellular processes and null or strong mutations cause cell lethality. In contrast, hypomorphic mutations do not significantly perturb Wg signaling, probably because a low dose of CKIε suffices to transduce the Wg signal and/or because other CKI family members can compensate for the partial loss of CKIε. To facilitate the recovery of mutant clones homozygous for dbt null mutation, a combination of several approaches was applied: (1) mitotic clones were generated in the Minute background, which gave mutant cells a growth advantage; (2) P35, a cell death inhibitor, was overexpressed in discs where dco mutant clones were generated to block apoptosis due to loss of CKIε; (3) a wing specific, constitutive source of flipase (MS1096/UAS-flp) was used to induce FRT-mediated mitotic recombination in the wing pouch region. Under these conditions, all wing discs of the appropriate genotype contained dco clones occupying most of the wing pouch region. These wing discs exhibited diminished levels of Wg target gene expression, demonstrating that DBT/CKIε is a positive regulator of the Wg pathway. The approach described in this study can be applied to study other cell lethal genes (Zhang, 2006).

Although most of the evidence supports a positive role for CKIε in the Wnt/Wg pathway, several observations implied that CKIε also impinged on β-catenin/Arm phosphorylation and degradation. For example, it has been shown that CKIε is associated with Axin and DN-CKIε blocks Axin-induced phosphorylation of β-catenin at Ser45. In addition, RNAi knockdown of DBT/CKIε resulted in stabilization of Arm in S2 cells, albeit to a lesser extent than CKIα knockdown, and increased the basal transcription from a Tcf-luciferase reporter gene. However, one caveat of these studies is that the activities of other CKI isoforms might also be affected by DN-CKIε or DBT/CKIε RNAi. A genetic approach was taken to address whether DBT/CKIε has any negative function in the Wg pathway, and hypomorphic dbt mutations were found to cause ectopic Wg signaling, but only when CKIα activity was partially blocked. Hence, DBT/CKIε is normally dispensable for Arm degradation due to sufficent CKIα; however, DBT/CKIε levels become critical when CKIα activity is reduced. This result is not inconsistent with a previous observation that CKIε RNAi did not affect β-catenin phosphorylation and degradation in cultured cells (Liu, 2002). In that study, RNAi did not completely block CKIε, and the presence of CKIα in the same cells could have masked any effect CKIε RNAi might have had on β-catenin phosphorylation and degradation. It would be interesting to determine if CKIε RNAi could enhance the effect of CKIα RNAi on β-catenin phosphorylation and degradation, which is predicted by the current study (Zhang, 2006).

CKIε binds and phosphorylates Dsh. However, a previous study placed CKIε downstream of Dsh based on the observation that overexpressing XCKIε could rescue Wnt signaling defects caused by a dominant negative form of Dsh (DN-Dsh). In contrast, this study found that the ability of XCKIε to induce Wg pathway activation depends on Dsh, as dsh null mutant clones overexpressing XCKIε fail to activate Wg target genes. Hence genetic epistasis study places CKIε upstream of or parallel to Dsh. It is possible that DN-Dsh might not completely block endogenous Dsh, and overexpressed XCKIε could transduce the Wnt signal through residual Dsh activity. Consistent with the notion that CKIε acts upstream of or parallel to Dsh, it was found that coexpression of Nkd, an inducible Wg pathway inhibitor that acts by binding to Dsh, suppresses the 'gain-of-Wg' phenotypes caused by XCKIε. In addition, DN-GSK3β can reverse the 'loss-of-Wg' phenotypes caused by DN-CKIε. Hence a critical role that CKIε plays is to antagonize the activity of the Arm/β-catenin destruction complex, and antagonism of GSK3β alleviates such a requirement. CKIε could bind Dsh and destabilize the Arm/β-catenin destruction complex. In addition, CKIε could destabilize Axin complex through phosphorylation of Arr (Zhang, 2006).

Epistasis analysis also revealed a role for CKIε downstream of GSK3β phosphorylation. It was found that the levels of ectopic sen in wing discs coexpressing DN-CKIε and DN-GSK3β are significantly lower than those in wing discs expressing DN-GSK3β alone, suggesting that DN-CKIε attenuates Wg signaling activity even when phosphorylation and degradation of Arm is blocked by DN-GSK3β. One likely target for CKIε downstream of GSK3β is Tcf as it has been shown that in Xenopus oocyte extracts, CKIε phosphorylated Tcf3 and stabilized its interaction with β-catenin (Zhang, 2006).

The role of CKIα in the Wnt/Wg pathway has largely been deduced from studies using cell culture systems. Thus, RNAi knockdown of CKIα inhibits β-catenin/Arm phosphorylation and degradation, and induces Tcf/Lef mediated luciferase expression. CKIα RNAi in Drosophila embryos resulted in a 'naked cuticle' phenotype, consistent with ectopic Wg signaling (Liu, 2002; Yanagawa, 2002). However, two recent studies revealed that loss-of-CKIα also results in ectopic Hh signaling. This finding complicated the interpretation of the 'gain-of-Wg' phenotypes resulting from CKIα RNAi as Hh and Wg regulate each other’s expression in Drosophila embryos. To circumvent this problem, this study used Drosophila wing development as a model to address the In vivo function of CKIα since Wg and Hh do not regulate each other in this system. It was found that overexpressing two shorter forms of CKIα RNAi constructs (CRS and CRS2), which are specific for CKIα, led to ectopic Wg signaling in a dose dependent manner: one copy of CRS or CRS2 barely affected Wg target gene expression whereas two copies resulted in ectopic expression of sc and sen. A longer form of CKIα RNAi construct (CRL) was more potent than CRS, as expressing one copy resulted in robust ectopic expression of sc and sen. This is likely due to the fact that CRL knocks down CKIα more effectively than CRS. In addition, CRL may knock down DBT/CKIε to reduce a compensatory effect on loss of CKIα by DBT/CKIε. Intriguingly, expressing CRL at higher levels caused adverse effect on the Wg signaling activity, as manifested by the reduced levels of ectopic sc expression. A likely explanation is that high levels of CRL diminish the level of CKIε to the extent that its positive role in the Wg pathway is compromised. In support of this notion, coexpressing DBT/CKIε with CRL restored high levels of ectopic sc expression (Zhang, 2006).

Despite the predominantly negative role of CKIα in the Wg pathway, a positive role has been underscored in double mutant analysis. It was observed that CKIα knockdown enhanced the 'loss-of-Wg' phenotypes caused by dbt null mutation, as manifested by more complete loss of sen and vg expression in dbt mutant discs expressing CRS2. CKIα may positively regulate Wg signaling by phosphorylating Dsh, as suggested by previous studies. Alternatively, CKIα could exert a positive influence on the Wg pathway by phosphorylating Arr (Zhang, 2006).

Overexpression assays were applied to explore the potential role of the other five CKI isoforms that share over 50% amino acid sequence identity in their kinase domains with CKIα. First, it was asked if any of these CKI isoforms could functionally substitute for CKIα in blocking Wg pathway activation. Unlike CKIα, none of other CKI isoforms including CG7094, CG2577, CG12147, Gish/CKIγ, and CG9962 were able to rescue the 'gain-of-Wg' phenotype caused by CRL, suggesting that these CKI isoforms are unlikely to play any major role in priming GSK3β-mediated phosphorylation and degradation of Arm/β-catenin. In contrast, CG12147 induced ectopic Wg signaling activity when CKIα was partially blocked, albeit to a lesser extent than DBT. Although Gish overexpression failed to induce ectopic Wg signaling activity even when CKIα was partially blocked, loss-of-Gish mutation resulted in a reduction in Wg signaling activity and enhanced the 'loss-of-Wg' phenotypes caused by the dbt null mutation, suggesting that Gish/CKIγ positively regulates the Wg pathway (Zhang, 2006).

It has recently been shown that CKI family members phosphorylate multiple sites in the cytoplasmic domain of LRP6 (Davidson, 2005; Zeng, 2005) and a set of these CKI sites are primed by GSK3β phosphorylation of the PPPSP motif (Zeng, 2005). Overexpressing CKIγ but not CKIε caused phosphorylation of LRP6, whereas dominant negative CKIγ inhibited Wnt3a-induced LRP6 phosphorylation in HEK293T cells (Davidson, 2005), suggesting a specific role for CKIγ in phosphorylating LRP6. In contrast, Zeng showed that a combination of dominant negative CKIα and CKIδ but neither CKIα or CKIδ alone blocked Wnt3a-induced LRP6 phosphorylation in CKIε−/− MEF cells, suggesting that CKIα and CKIγ/ε act redundantly in phosphorylating LRP6 in response to Wnt (Zeng, 2005). However, dominant negative CKI isoforms may not exhibit absolute specificity, which could account for the discrepancy between these two studies. While it awaits for genetic mutations in individual CKI isoforms to confirm the results obtained with the dominant negative forms of CKI, it is likely that multiple CKI family members could participate in LRP5/6 phosphorylation (Zhang, 2006).

Multiple PPPSP motifs as well as adjacent CKI sites are conserved in the cytoplasmic domain of Drosophila Arr. In Drosophila S2 cells, multiple CKI family members can phosphorylate Arr cytoplasmic domain and this phosphorylation appears to rely on GSK3β primed phosphorylation. Among all the CKI isoforms that can phosphorylate Arr, Gish/CKIγ exhibited the highest potency whereas CKIα and CKIε show weak activity toward Arr, suggesting that Gish/CKIγ is the major CKI isoform that phosphorylates Arr. Consistent with its high potency toward Arr phosphorylation, Gish/CKIγ is primarily associated with plasma membrane, as is the case for its vertebrate counterpart (Davidson, 2005). Phosphorylation of Arr by Gish/CKIγ is likely to account for the positive role that Gish/CKIγ plays in the Wg signaling pathway. It was found that gishe01759 attenuates but not completely blocks Wg responsive gene expression. The residual Wg signaling activity in gishe01759 mutant cells could be due to the hypomorphic nature of this mutation. Alternatively, other CKI isoforms could partially substitute for Gish/CKIγ in phosphorylating Arr (Zhang, 2006).

CG12147 and CG9962 phosphorylate Arr more effectively than CKIα or CKIε, although they are less potent than Gish/CKIγ. Consistent with their ability to phosphorylate Arr, overexpressing CG12147 or CG9962 resulted in ectopic Wg signaling in a genetic sensitized background. However, phosphorylation of Arr alone might be insufficient to account for their positive roles as overexpressing Gish/CKIγ did not have the same magnitude of effect on Wg signaling as CG12147 and CG9962. It is possible that CG12147 and CG9962 can phosphorylate other targets in the Wg pathway. Future loss of function study and biochemical analysis should probe the precise roles of these CKI isoforms in the Wg pathway (Zhang, 2006).

Cell cycle control of Wnt receptor activation

Low-density lipoprotein receptor related proteins 5 and 6 (LRP5/6; Drosophila Arrow) are transmembrane receptors that initiate Wnt/β-catenin signaling. Phosphorylation of PPPSP motifs in the LRP6 cytoplasmic domain is crucial for signal transduction. Using a kinome-wide RNAi screen, it was shown that PPPSP phosphorylation requires the Drosophila Cyclin-dependent kinase (CDK) L63. L63 and its vertebrate homolog PFTK are regulated by the membrane tethered G2/M Cyclin, Cyclin Y, which mediates binding to and phosphorylation of LRP6. As a consequence, LRP6 phosphorylation and Wnt/β-catenin signaling are under cell cycle control and peak at G2/M phase; knockdown of the mitotic regulator CDC25/string, which results in G2/M arrest, enhances Wnt signaling in a Cyclin Y-dependent manner. In Xenopus embryos, Cyclin Y is required in vivo for LRP6 phosphorylation, maternal Wnt signaling, and Wnt-dependent anteroposterior embryonic patterning. G2/M priming of LRP6 by a Cyclin/CDK complex introduces an unexpected new layer of regulation of Wnt signaling (Davidson, 2009).

Wnt/β-catenin signaling regulates patterning and cell proliferation throughout embryonic development and is widely implicated in human disease, notably cancer. Two principal classes of transmembrane (TM) receptors function to transduce Wnt/β-catenin signaling; the seven pass TM Frizzled (Fz) proteins and the single pass TM low density lipoprotein receptor-related proteins 5 and 6 (LRP5/6; Drosophila Arrow). Frizzled receptors activate β-catenin-dependent (canonical) as well as β-catenin-independent (noncanonical, such as planar cell polarity) pathways, while LRP5/6 function more specifically in the Wnt/β-catenin pathway (Davidson, 2009).

LRP6 signaling requires Ser/Thr phosphorylation of its intracellular domain (ICD), which contains five PPPSPXS dual phosphorylation motifs comprising Pro-Pro-Pro-Ser-Pro (PPPSP) and directly adjacent casein kinase 1 (CK1) sites. Phosphorylation of the most N-terminal PPPSP (S1490) involves glycogen synthase kinase 3 (GSK3), while CK1g phosphorylates two Ser/Thr clusters near S1490. Phosphorylation of CK1 sites is downstream of, and requires, PPPSP phosphorylation; however, alternative epistasis models have also been proposed. Both PPPSP and CK1 site phosphorylation is necessary for Axin binding to LRP6 and Wnt/β-catenin pathway activation. Phosphorylated PPPSPXS motifs directly inhibit the ability of GSK3 to phosphorylate β-catenin, providing a potential mechanism linking LRP6 activation to β-catenin stabilization. Investigating how LRP6 phosphorylation is regulated is thus crucial for understanding Wnt receptor activation and downstream signaling. Constitutive, non-Wnt-induced S1490 phosphorylation has been observed, suggesting that additional proline-directed kinases may be involved, such as the ERK or Cyclin-dependent kinase (CDK) subgroups (Davidson, 2009).

CDKs are regulators of the cell cycle and require Cyclin partners, whose levels are precisely controlled during the cell cycle, endowing CDKs with both temporal activity and substrate specificity. Several less well-characterized CDK-like proteins exist, including the PFTAIRE kinase subfamily. This study reports on the identification of a Cyclin/PFTAIRE-CDK complex that phosphorylates LRP6 S1490 in a cell cycle-dependent manner, which brings Wnt/β-catenin signaling under G2/M control and introduces a surprising new principle in Wnt regulation (Davidson, 2009).

An important issue in the field of Wnt/β-catenin signaling concerns the regulation of LRP5/6/Arrow function via phosphorylation. This study has identified the unusual plasma membrane tethered Cyclin Y/PFTAIRE complex which functions predominantly at the G2/M phase of the cell cycle to phosphorylate the PPPSP motifs of LRP6. The results suggest a G2/M priming model of LRP5/6/Arrow phosphorylation, where the Cyclin Y/CDK complex phosphorylates LRP6 at PPPSP motifs, which then primes adjacent phosphorylation by CK1. However, PPPSP priming alone is not sufficient for phosphorylation by CK1, as Wnt-induced LRP6 aggregation is also required. Combined phosphorylation at PPPSP and CK1 sites then promotes Gsk3-Axin binding to LRP6 and signalosome formation. Since GSK3 and Cyclin Y/CDK are both essential for LRP6 priming they apparently act nonredundantly. So why is there a dual kinase input to PPPSP phosphorylation? The phosphorylation of LRP6 by GSK3 occurs in acute response to Wnt signaling and it was suggested that it serves to amplify receptor activation. Cyclin Y/CDK phosphorylates Wnt independently at G2/M, thereby gating signal transduction in proliferating cells. One possibility is that individually both kinases prime LRP6 substoichiometrically at the five PPPSP sites and that only their combined action is sufficient for full LRP6 signaling competence (Davidson, 2009).

These findings have important implications for the link between proliferation and Wnt signaling. It has been long known that there is cross talk between mitogenic growth factors and Wnt signaling. The current results may explain why mitogenic growth factors synergize with Wnt/β-catenin signaling, namely by G2/M priming of LRP6 through enhanced cell proliferation, which sensitizes LRP6 for incoming Wnt signals. Moreover, not only extracellular but also intracellular cell cycle check point regulators controlling G2/M entry are likely to affect Wnt signaling (Davidson, 2009).

Wnt/β-catenin signaling itself promotes G1 progression by inducing c-myc and cyclin D1. This suggests that Wnt/β-catenin signaling can entrain a positive feedback loop in proliferating cells by promoting cell cycle progression, which triggers LRP6 phosphorylation at G2/M. Simultaneous stimulation by Wnt and mitogenic growth factors could initiate such a loop. Indeed, the results may explain the previously noted G2/M enrichment of β-catenin and Wnt signaling. Likewise, protein levels of the direct Wnt target gene Axin2, considered a marker gene for Wnt/β-catenin signaling, also peak during mitosis (Davidson, 2009).

What may be the function of a Wnt positive feedback loop during the cell cycle? One of the many roles of Wnt/beta-catenin signaling is to promote cell proliferation and the positive feedback loop suggested by this study may enhance the systems' levels properties of the cell cycle. Specifically, the loop may promote synchrony of cell cycle regulated events or constitute a bistable switch between cell proliferation and cell cycle exit (Davidson, 2009).

One interesting question raised by this study concerns preferential transcription of Wnt target genes around G2/M. Most genes are transcriptionally silenced between late prophase and early telophase, yet TOPFLASH reporter and AXIN2 peak around G2/M. It will therefore be interesting to investigate whether Wnt target genes are transcribed during the more permissive stages G2, early prophase, or late telophase (Davidson, 2009).

Another important question raised by this study is whether G2/M priming is essential or only modulatory for Wnt/β-catenin signaling in general, in particular in light of Wnt signaling in nondividing cells. The fact that LRP6 signaling is promoted by G2/M phase does not exclude Wnt/β-catenin signaling in other cell cycle phases or in nondividing cells. Even though during interphase the levels of LRP6 signalosomes, Sp1490, β-catenin, and reporter activation are lower compared to G2/M, such Wnt/β-catenin signaling is likely physiologically relevant and may involve additional PPPSP kinases, such as GSK3. Surprisingly little is known about Wnt/β-catenin signaling in nondividing cells. In transgenic Wnt-reporter mice, Wnt activity is detected in apparently postmitotic cells in the adult brain, retina, and certain liver cells. In the adult liver, Wnt/β-catenin signaling controls perivenous gene expression. Furthermore, Wnts play a role in axon remodeling in postmitotic neurons and at least one study suggests that this can involve the β-catenin pathway. In light of the current results it will be interesting to examine more systematically Wnt/β-catenin signaling and in particular the LRP6 kinases involved in postmitotic cells (Davidson, 2009).

Traditionally it is thought that Wnt/β-catenin signaling acts to regulate gene expression of downstream targets. Why then should Wnt/β-catenin signaling peak at G2/M? One likely answer is that components of the Wnt/β-catenin pathway play a crucial role during mitosis beyond transcriptional activation. In C. elegans, Wnt signaling regulates the orientation of the mitotic spindle in early development. In mammalian cells, phosphorylated β-catenin itself binds to centrosomes and is involved in spindle separation during mitosis. Likewise, GSK3, Adenomatous polyposis coli protein (APC) and Axin2, which are components of the β-catenin destruction complex, also have direct functions in mitosis. Taken together these data suggest that Cyclin Y/CDK phosphorylates LRP6 at G2/M to induce Wnt/β-catenin signaling for orchestrating a mitotic program (Davidson, 2009).


The arrow gene was cloned using a lethal P-element insertion [l(2)k08131] that fails to complement arrow mutants. The longest complementary DNA (6,012 base pairs) restores smooth cuticle to alternate segments of arrnull embryos when expressed under control of Prd-GAL4, indicating rescue of Arrow function and Wg signaling (Wehrli, 2000).

Initially, Arrow messenger RNA is distributed uniformly in the embryo, owing to maternal contribution, but by stage 9 broad stripes are superimposed on this global expression. As zygotic expression takes over, Arrow mRNA stripes become more accentuated, such that by stage 13 the highest levels are posterior to the En domain, and the lowest levels are just anterior to the En domain, in the region signaled by Wg at this stage. Similarly, in leg discs arrow transcription is lowest in cells expressing Wg. This suggests that arrow transcription is negatively regulated by Wg signaling, in a manner similar to the Frizzled receptors Fz and DFz2, but unlike the signal transducers Dsh and Arm, which are both uniformly expressed. Although the significance of this transcriptional regulation is unclear, it is unlikely that it is relevant to signaling in the embryo, as global maternal arrow contribution suffices for most epidermal Wg signaling events. Polymerase chain reaction with reverse transcription (RT-PCR) analysis shows also that arrow mRNA is expressed in S2 cells, consistent with these cells being able to respond to Wg once transfected with DFz2 (Wehrli, 2000).

Effects of Mutation or Deletion

The Drosophila retina is made from hundreds of asymmetric subunit ommatidia arranged in a crystalline-like array, with each unit shaped and oriented in a precise way. One explanation for the precise cellular arrangements and orientations of the ommatidia is that they respond to two axes of polarized information present in the plane of the retinal epithelium. Earlier work has shown that one of these axes lies in the anterior/posterior(A/P) direction and that the polarizing influence is closely associated with the sweep of the Hedgehog-dependent morphogenetic wave. Evidence is presented for a second and orthogonal axis of polarity: this signal can be functionally separated from the A/P axis (see Specification of the eye disc primordium and establishment of dorsal/ventral asymmetry). The polarizing information acting in this equatorial/polar axis (Eq/Pl) is established in at least two steps -- the activity of one signaling molecule functions to establish the graded activity of a second signal. Ectopic Wg expression results in two significant effects. (1) Clones are generated with associated polarity inversions. (2) Although significant changes in retinal polarity are associated with the clones, the distance over which the effect is exerted is restricted to from between 7 to 2 ommatidial rows. Ectopic Wg clones have two distinct features with respect to their polarity effects: (1) the aberrant polarity is asymmetrically distributed in relation to the clone (greater changes in polarity occur in polar positions relative to the center of the clone), and (2) the potency of the Wg-expressing clones to induce polarity reversals show maximial polarity-reversal effects at the equator and minimal effects at the pole (Wehrli, 1998).

Other genes downstream of wingless also appear associated with eye Eq/Pl polarity. The product of the arrow (arr) gene has been placed in the Wingless pathway based on a number of criteria:

To a variable extent, clones of armadillo and dishevelled induce polarity inversions on their equatorial side. The critical observation is that mutations in these recognized transducers of the Wg signal induce non-autonomus effects, consistent with their regulating the activity of a sendary signaling factor. This secondary signal is termed factor-X. Not only do arr, arm and dsh clones specifically affect the equatorial side, they are also more potent in achieving this at the pole than the equator. Thus it is inferred that factor-X activity is graded in the Eq/Pl axis but there is insufficient information to determine whether the activity is high at the equator and low at the poles, or vice-versa (Wehrli, 1998).

The abdomen of adult Drosophila consists of a chain of alternating anterior (A) and posterior (P) compartments which are themselves subdivided into stripes of different types of cuticle. Most of the cuticle is decorated with hairs and bristles that point posteriorly, indicating the planar polarity of the cells. This study has focused on a link between pattern and polarity. Previous studies have shown that the pattern of the A compartment depends on the local concentration (the scalar) of a Hedgehog morphogen produced by cells in the P compartment. Evidence is presented in this study that the P compartment is patterned by another morphogen, Wingless, which is induced by Hedgehog in A compartment cells and then spreads back into the P compartment. Both Hedgehog and Wingless appear to specify pattern by activating the optomotor blind gene, which encodes a transcription factor. A working model that planar polarity is determined by the cells reading the gradient in concentration (the vector) of a morphogen 'X' which is produced on receipt of Hedgehog, is re-examined. Evidence is presented that Hedgehog induces X production by driving optomotor blind expression. X has not yet been identified and data is presented that X is not likely to operate through the conventional Notch, Decapentaplegic, EGF or FGF transduction pathways, or to encode a Wnt. However, it is argued that Wingless may act to enhance the production or organize the distribution of X. A simple model that accommodates these results is that X forms a monotonic gradient extending from the back of the A compartment to the front of the P compartment in the next segment, a unit constituting a parasegment (Lawrence, 2002).

In clones mutant for arm or arrow, the expectation was that the Wg pathway in these two types of clones would be blocked. Two effects were noted. (1)The clones in the dorsal epidermis differentiated cuticle characteristic of the ventral epidermis: they made pleural hairs, and patches of sternite. Clones in all portions of the tergite, in both the A and P compartments, were transformed in this manner, indicating a general requirement for Wnt signaling to specify dorsal as opposed to ventral structures. Thus, in the wild type, all dorsal cells are probably exposed to at least low levels of Wg or some other Wnt protein. (2) Such clones affect polarity: in the tergites, the mutant clones were normal at the rear of the clone but reversed in the front, with reversal extending outside the clone. One explanation for these polarity changes could be that, in the tergites, Wg normally acts to enhance the production of X. Thus cells deficient in the Wnt pathway would produce less X than normal, giving a dip in the concentration landscape for X, causing reversed polarity at the front of the clone. In the eye, both arm- and arrow- clones cause equivalent polarity reversals and a similar resolution has been offered: it is suggested that Wg might regulate the production of a secondary polarizing factor also dubbed X (Lawrence, 2002).

Thus, it is proposed that Wg helps to produce X, but that Wg itself is not X. If Wg were X, both arm- and arrow- clones should not be able to transduce it, and hence, should have random polarity within the clone. Moreover, the effects on polarity should be cell autonomous. Yet, as has been seen, these clones behave as if they have caused an altered distribution of X, rather than any failure to transduce X. Similar arguments apply to sgg- clones. In this case, the Wg pathway should be constitutively activated in all cells within the clone, preventing them from detecting a gradient of Wg protein. However such clones are not randomly polarized, indicating that they can still respond to graded X activity (Lawrence, 2002).

It is useful to compare the roles of Omb and Wg on X production. Omb is apparently essential for X production: omb- clones at the back of A show reversed polarity that extends all the way to the posterior edge of the compartment. By contrast, in arm- and arrow- clones, reversal occurs only in the anterior portions of such clones. Thus, it is inferred that arm- and arrow- cells located at the back of A can produce some X, even though they cannot activate the canonical Wnt pathway. Thus, it could be that Hh drives X production mainly through Omb, but also adds to the level of X produced through the induction and action of Wg. The combination of both Omb and Wg activity might extend the reach of the X gradient to encompass the whole A compartment, and possibly also further forward into the neighboring P compartment (Lawrence, 2002).

None of the previous studies has helped gain an understanding of how the P compartment is patterned or how its cells are polarized. smo- clones have no phenotype in the P compartment, confirming that Hh has no function there. In the embryo and imaginal discs, Hh crossing over from the P compartment induces the expression of Wg and Dpp in line sources along the back of A. Both proteins then spread back into the P compartment where they act as gradient morphogens to control P growth and pattern. Wg and Dpp are also produced at the back of the A compartment in each abdominal segment (albeit in distinct dorsal and ventral domains). Hence, by analogy with the embryo and imaginal discs, these morphogens seem to be the most likely candidates to pattern the P compartment here as well. If so, it would be supposed that in the tergites, Hh induces Wg and this Wg moves posteriorly across the AP compartment boundary into the P compartment where it activates expression of omb, thus specifying the zone of hairy cuticle (p3) and distinguishing it from p2 cuticle, which is bald. This hypothesis was tested in the following experiments (Lawrence, 2002).

If Wg activates omb in anterior regions of the P compartment, blocking the Wnt pathway in cells in the P compartment should block expression of omb. Expression of omb was therefore monitored in arrow- clones. omb is sometimes, but not always, turned off autonomously in the clone. Conversely, ectopic activation of the Wnt pathway should transform bald cuticle (p2) at the back of P into hairy cuticle (p3) normally found at the front of P. Indeed, some clones lacking the sgg gene become hairy if situated in the bald areas of P, apparently causing a transformation from p2 to p3 cuticle. But, clones expressing either tethered Wg or activated Arm, which should behave similarly, have no clear effects. Even so the positive results with arrow and sgg give support to the hypothesis that Wg stratifies the P compartment by working through Omb (Lawrence, 2002).

Canonical Wnt signaling in the visceral muscle is required for left-right asymmetric development of the Drosophila midgut

Many animals develop left-right (LR) asymmetry in their internal organs. The mechanisms of LR asymmetric development are evolutionarily divergent, and are poorly understood in invertebrates. Drosophila has several organs that show directional and stereotypic LR asymmetry, including the embryonic gut, which is the first organ to develop LR asymmetry during Drosophila development. This study found that genes encoding components of the Wnt-signaling pathway are required for LR asymmetric development of the anterior part of the embryonic midgut (AMG). frizzled 2 and Wnt4, which encode a receptor and ligand of Wnt signaling respectively, are required for the LR asymmetric development of the AMG. arrow, an ortholog of the mammalian gene encoding low-density lipoprotein receptor-related protein 5/6, which is a co-receptor of the Wnt-signaling pathway, was also essential for LR asymmetric development of the AMG. These results are the first demonstration that Wnt signaling contributes to LR asymmetric development in invertebrates, as it does in vertebrates. The AMG consists of visceral muscle and an epithelial tube. Genetic analyses revealed that Wnt signaling in the visceral muscle but not the epithelium of the midgut is required for the AMG to develop its normal laterality. Furthermore, fz2 and Wnt4 are expressed in the visceral muscles of the midgut. Consistent with these results, it was observed that the LR asymmetric rearrangement of the visceral muscle cells, the first visible asymmetry of the developing AMG, did not occur in embryos lacking Wnt4 expression. These results also suggest that canonical Wnt/β-catenin signaling, but not non-canonical Wnt signaling, is responsible for the LR asymmetric development of the AMG. Canonical Wnt/β-catenin signaling is reported to have important roles in LR asymmetric development in zebrafish. Thus, the contribution of canonical Wnt/β-catenin signaling to LR asymmetric development may be an evolutionarily conserved feature between vertebrates and invertebrates (Kuroda, 2012).

This study found that Wnt-signaling components Wnt4 and Fz2 are required for LR asymmetric development of the AMG, although contribution of other Wnt ligands and receptors to this process could not be excluded. For example, it is known that Wnt4 binds to Fz and Fz2, and that fz and fz2 function redundantly in the segmentation of Drosophila embryos. This study found that the AMG of embryos homozygous for fz showed similar LR defects to those of fz2, although at a lower frequency. Therefore, it is possible that Fz acts redundantly as the receptor for canonical Wnt/β-catenin signaling, although the expression of fz in the midgut could not be detected by anti-Fz antibody staining. In contrast, analysis of embryos homozygous for derailed (drl) suggested that Wnt5 may not be involved in the LR asymmetric development of the AMG. Drl is a member of the RYK subfamily of receptor tyrosine kinases and is a receptor for Wnt5. The laterality of the AMG was normal in embryos homozygous for drl (Kuroda, 2012).

Wnt4 is one of the few Wnt ligands whose function has been revealed in Drosophila. This study found that Wnt4–Fz2 activates the canonical Wnt/β-catenin signaling pathway for normal LR asymmetric development of the AMG. Consistent with this finding, Wnt4 activates the canonical Wnt/β-catenin signaling pathway in salivary glands through Fz or Fz2, which is required for the glands’ proper migration. However, the Wnt4–Fz2 pathway is also known to activate non-canonical Wnt signaling in other systems. Wnt4 plays an essential role in the cell movement required for formation of the ovariolar sheath cells. In addition, Wnt4 expressed in the developing ventral lamina is required for ventral projection of the retinal axon. In both of these cases, Fz2 acts as a receptor of Wnt4, and the Wnt4–Fz2 pathway activates non-canonical Wnt signaling. Therefore, although the same combination of Wnt ligand and receptor, Wnt4–Fz2, is involved, the downstream cascades of Wnt signaling may be context-dependent, although the factors acting as molecular switches for these downstream pathways remain unknown (Kuroda, 2012).

The first indication of LR symmetric morphogenesis in the AMG is observed as the LR asymmetric rearrangement of circular visceral muscle (CVMU) cells. These rearrangements can be monitored by measuring the major axial angle of the nuclei in the CVMU cells to the midline of the AMG (Kuroda, 2012).

This study found that the LR asymmetry of the rearranged CVMU cells in the ventral AMG became bilaterally symmetric in embryos homozygous for a Wnt4 mutation. This result was consistent with the AMG’s random LR laterality in these embryos. However, unexpectedly, the CVMU cells were rearranged LR asymmetrically in the dorsal AMG in Wnt4 mutant homozygotes, even though the arrangement of these dorsal cells is bilaterally symmetric in wild-type embryos. This result suggests that Wnt signaling may counteract the LR asymmetric morphogenesis in the dorsal side of the AMG, in addition to its role in introducing a LR bias by inducing the rearrangement of CVMU cells in the ventral AMG, via the Wnt4–Fz2 pathway. In embryos homozygous for loss-of-function mutations of Wnt4, arr, or fz2, the LR asymmetric development of the posterior embryonic gut was largely normal. Thus, in wild-type embryos, the Wnt4–Fz2 signal may function to suppress the influence of the LR asymmetric morphogenic signals from the posterior midgut on the AMG (Kuroda, 2012).

The present analyses clarified the requirement for Wnt4–Fz2 signaling in the LR asymmetric morphogenesis of the AMG, but the precise molecular functions of this signal are still unclear. Because Wnt4–Fz2 activates canonical Wnt/β-catenin signaling, it will be important to identify the target genes responsible for LR asymmetric morphogenesis of the AMG (Kuroda, 2012).

Wnt signaling is required for long-term memory formation

Wnt signaling regulates synaptic plasticity and neurogenesis in the adult nervous system, suggesting a potential role in behavioral processes. This study probed the requirement for Wnt signaling during olfactory memory formation in Drosophila using an inducible RNAi approach. Interfering with β-catenin expression in adult mushroom body neurons specifically impairs long-term memory (LTM) without altering short-term memory. The impairment is reversible, being rescued by expression of a wild-type β-catenin transgene, and correlates with disruption of a cellular LTM trace. Inhibition of wingless, a Wnt ligand, and arrow, a Wnt coreceptor, also impairs LTM. Wingless expression in wild-type flies is transiently elevated in the brain after LTM conditioning. Thus, inhibiting three key components of the Wnt signaling pathway in adult mushroom bodies impairs LTM, indicating that this pathway mechanistically underlies this specific form of memory (Tan, 2013).

This study was prompted by a previous discovery that a casein kinase Iγ homolog (CkIγ), gilgamesh (gish), is required for STM in Drosophila (Tan, 2010). CkIgγmediated phosphorylation of the cytoplasmic tail of Lrp5/6 (Arr) is crucial for Wnt/β-catenin signaling (Davidson, 2005), and it was predicted that disruption of the Wnt signaling pathway would perturb STM. Surprisingly, however, it was found that knockdown of the four Wnt signaling components leaves STM intact. The likely explanation for this discrepancy is that Gish serves other important functions in STM formation besides its role in LTM through phosphorylation of the Arr receptor (Tan, 2013).

How does Wnt signaling in the MB neurons mediate the formation of LTM? Since the normal expression of β-catenin, Wg, and Arr is required in the set of MB neurons defined by P{MB-GeneSwitch}12-1, and Wg is a short-range ligand, a model is favored in which the Wnt ligand, Wg, participates in an autocrine fashion in the MB neurons. Spaced conditioning, which produces long-term behavioral memory, but not massed or single-cycle conditioning, leads to a transient increase in wg expression in the MB neurons, perhaps as a step downstream of Creb. The subsequent secretion of Wg by the MB neurons activates the Fz/Arr receptor, leading to the accumulation of β-catenin in the MB neurons. β-catenin, in turn, orchestrates transcriptional changes in the MB neurons that are required for LTM, as well as the breaking and remaking of cell contacts through N-cadherin function, which is necessary for the reorganization of synapses for LTM storage. Recently, ribonucleoprotein particles containing synaptic protein transcripts were shown to exit the nucleus through a nuclear envelope budding process in response to Wnt signaling at the Drosophila neuromuscular junction (Speese, 2012). Wnt-dependent nuclear budding could provide the initial step for transporting RNAs to synapses for local protein synthesis and LTM formation (Tan, 2013).


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date revised: 18 June 2017

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